Sequence variation in ligand binding sites in proteins

BMC Bioinformatics20056:240

DOI: 10.1186/1471-2105-6-240

Received: 15 March 2005

Accepted: 30 September 2005

Published: 30 September 2005

The recent explosion in the availability of complete genome sequences has led to the cataloging of tens of thousands of new proteins and putative proteins. Many of these proteins can be structurally or functionally categorized from sequence conservation alone. In contrast, little attention has been given to the meaning of poorly-conserved sites in families of proteins, which are typically assumed to be of little structural or functional importance.

Recently, using statistical free energy analysis of tetratricopeptide repeat (TPR) domains, we observed that positions in contact with peptide ligands are more variable than surface positions in general. Here we show that statistical analysis of TPRs, ankyrin repeats, Cys2His2 zinc fingers and PDZ domains accurately identifies specificity-determining positions by their sequence variation. Sequence variation is measured as deviation from a neutral reference state, and we present probabilistic and information theory formalisms that improve upon recently suggested methods such as statistical free energies and sequence entropies.

Sequence variation has been used to identify functionally-important residues in four selected protein families. With TPRs and ankyrin repeats, protein families that bind highly diverse ligands, the effect is so pronounced that sequence "hypervariation" alone can be used to predict ligand binding sites.

The central challenge of the post-genomic era is to determine the structures and functions of thousands of newly-identified putative proteins [1, 2]. Elucidating how proteins carry out their functions in diverse contexts and in organisms from all three domains of life is both fundamentally important to understanding biological function and critical for engineering new functions into novel proteins. Sequence conservation alone can be used to structurally categorize many proteins or putative proteins [3]. Additionally, catalytic sites in enzymes can sometimes be identified from conserved surface motifs [48]. In contrast, sites with poor sequence conservation have been largely ignored, because they are assumed to be of little structural or functional importance [9].

Sequence alignment of proteins with similar structures has shown that as sequence identity increases, average backbone variation decreases [10]. Within a particular protein family, residues in the hydrophobic core are the most conserved, implying they play a key role in specifying the fold [11]. In contrast, solvent-exposed residues tend to be variable, with mutations having little deleterious effect on overall structure or stability [12]. Consequently, conservation of surface residues is commonly taken to be an indication of functional importance [13]. This idea can be used to identify active-site residues from a collection of proteins that perform the same function, but it is not applicable to families of proteins that use a common scaffold to bind diverse ligands. Rather, we might hypothesize that such binding sites will be composed of positions that are variable.

Recently, we used a statistical free energy (SFE) approach [14, 15] to understand better the role of conserved residues in defining tetratricopeptide repeat (TPR) motifs, which are thought to commonly mediate protein-protein interactions [16]. Strikingly, we found that the ligand-binding site of the motif can readily be identified by sequence hypervariation of positions proximal to the ligand, as evidenced by very low statistical free energies separating those positions from a position-independent reference state. Here, we examine this observation in more detail and demonstrate that specificity-determining residues in TPRs, ankyrin (Ank) repeats, Cys2His2 zinc fingers (Zifs), and PDZ domains can be identified from sequence variation.

By analyzing protein families with exceptional biochemical and biophysical characterization, we show that, when the ligand repertoire is highly diverse for a particular family, the binding site can be identified from sequence hypervariation alone. However, even when the ligands have significant features in common, sequence variation can be used to "dissect" binding sites to identify specificity-determining residues. We demonstrate this sequence variation using probabilistic and information theory approaches closely tied to the mathematics of covariation, which are more suitable for this type of analysis than SFEs or Shannon entropies. Statistical identification of specificity-determining residues will greatly facilitate the engineering proteins with novel functions and targets.

Results & discussion

The TPR binding site

The TPR is a common 34 amino-acid protein motif that occurs in arrays, most frequently with three contiguous repeats [17]. Although TPR domains are thought to mediate protein-protein interactions, only a few examples have been well characterized. The large number of known TPR sequences, nearly 10,000 in Pfam [18], makes this motif an excellent target for statistical analysis. Using several mathematical approaches, we calculated the separation of the amino acid distribution at each position in TPRs from a position-independent reference state, amino acid usage in all proteins in yeast (Figure 1a). Note that we have performed this calculation on all of the 34 positions in the TPR motif; TPR domains are made up of tandem repeats of the TPR motif.
Figure 1

Relative entropy analysis of 6,887 canonical-length (34 aa) TPR repeats. (a) The relative entropy values are shown for each TPR position, with secondary structure indicated (cylinders represent helices and lines represent loops). Arrows indicate the positions of the seven most variable residues. These values are mapped onto the co-crystal structures of HOP-TPR1/Hsc70 peptide (b) and HOP-TPR2A/Hsp90 peptide (c), with the TPR domains rendered in spheres and the ligands in sticks. Two views from 180° rotation of each molecule are shown. The concave, ligand binding surfaces, left, are clearly more variable than the convex, solvent exposed surfaces, right. A small insertion in TPR2A is colored grey. (d) Views of the concave binding surfaces as in (c), but only those residues known to contact the ligand from co-crystal structures are colored [19]. Rendered from PDB entries 1ELW and 1ELR using PyMOL.

When relative entropy values are mapped onto the ligand-bound co-crystal structures of two different three-TPR domains (the TPR1 and TPR2A domains from Hsp-Organizing Protein, HOP [19]), it is immediately apparent that the concave, peptide-binding face of the TPR domains is more variable (i.e., more like the reference state) than the convex, solvent-exposed face (Figure 1b and 1c). For clarity, the concave surfaces in Figure 1d are depicted with coloration of only ligand-binding residues independently identified from crystallographic analysis [19]. For both TPR domains, every residue in contact with the ligand peptide is in a position with a small relative entropy (blue or green in the figures), indicating small differences from the reference distribution.

This hypervariation is a consequence of each TPR having evolved to bind a different specific ligand (or portion of a ligand). When TPR proteins are considered collectively, the binding positions are statistically randomized to an extent that is dictated by the repertoire of amino acids required to perform the range of binding functions. In contrast, solvent-exposed residues in general mutate in a stochastic fashion throughout evolution, slowly reverting or "drifting" toward a "neutral" distribution. The lack of structural or functional importance of solvent-exposed residues results in little selective pressure against mutation, but the extent of randomization is limited by evolutionary time and subtle factors such as overall protein solubility. As a result of the high diversity of the ligands of TPR repeats, the binding surface is more variable than the solvent-exposed surface. Specifically, we predict that the ligand-binding residues that show the most sequence variation are the positions that determine ligand specificity.

Positions 2, 5, 9, 12, 13, 33 and 34 show the most sequence variation in TPRs (i.e., they have the lowest relative entropies, ≤ 0.30). These seven residues all lie on the same face of the motif, and they are the residues that are exposed on the concave face of TPR domains. In fact, the TPR-peptide co-crystal structures show that residues in positions 2, 5, 6, 9, 12, and 13 are used by HOP TPR1 and TPR2A to bind their ligands [19]. Since few TPR-ligand structures have been solved, it remains to be seen whether or not other TPR domains utilize positions 33 and 34. Their spatial proximity to the other binding residues suggests that this is likely.

It is not surprising that some of the positions in contact with the ligand peptides are more biased than others (such as position 6). Some positions used for binding may also have other restrictions (such as structural restrictions) that limit the repertoire of amino acids allowed, and some positions may be important for binding affinity but not for specificity (which is to say they may bind a feature that is common among all ligands). For example, position 6 is modestly conserved in TPRs overall (it is frequently Asn) and is more buried than the other binding positions. The position-6 residues make contacts to the backbone of the ligand peptides here. It is worth noting that a position-6 Asn in the PEX5 C-terminal TPR domain also appears to make contact to the peptide backbone of the unrelated peroxisomal targeting signal peptide [20].

Several of the spheres in TPR2A are grey, indicating that they correspond to non-canonical positions and were therefore not calculated. At present, our analysis does not consider the effect of insertions and deletions. In the future, one could imagine including "deletion" as another "amino acid," so that site occupancy would contribute to the variation score.

Measuring sequence variation

The use of metrics that measure the difference from a position-independent reference distribution is key to our observation, because (1) it is not clear how mere lack of conservation is related to variability, and (2) the likelihood of mutation from one residue to another is affected by factors such as genetic code bias and the greater difficulty of accommodating bulky or reactive amino acids. Here, we take the reference state to be amino acid usage in all open reading frames in Saccharomyces cerevisiae [21], which is independent of position but accounts for genetic code bias and amino acid chemistry. Using SFE calculations, we have previously demonstrated that using this reference state gives virtually indistinguishable results from other position-independent reference states such as amino acid usage in all proteins in the Pfam database [16].

We originally noted that the ligand binding site of TPRs was identified by sequence variation using statistical free energies [16]. SFEs are essentially a measure of the difference between amino acid distributions, relating the "probability" of observing a particular distribution to thermodynamic importance based on the exponential relationship given by the Boltzmann law.

(See Methods for an explanation of kT*.) This approach was introduced by Lockless & Ranganathan; however, their formalism for SFEs does not explicitly calculate the probability of observing a particular positional distribution [14]. Instead it uses the root-mean-square of the binomial probabilities of observing each amino acid, over all twenty amino acids x. That is,

where P x is given by

Here, N is the total number of sequences, n x is the number of sequences with amino acid x at the given position, and f x is the expected frequency of x from the reference state. This "vector" formalism for estimating the overall probability is empirically quite effective, but we speculated that a metric more tied to the mathematics of covariation would be more rigorous for our approach.

Since both of the following are true,

the probability of observing a particular distribution is simply given by the multinomial probability,
As expected, the ΔG stat values are very closely related (R2 = 0.89) to the ln P mult values for the 34 positions in the TPR motif (Figure 2a). Note, however, that the values of ΔG stat and ln P mult are dependent upon the total number of sequences N (since it is much less likely to observe a particular amino acid 200 times out of 1000 than 20 out of 100, if you are expecting it only 5% of the time).
Figure 2

Measuring differences in distributions. (a) Lockless & Ranganathan statistical free energies versus the logarithm of the multinomial probability for each of the 34 sites in TPRs. (b) Relationship of SFEs to sequence (Shannon) entropy for TPR sites. (c) Relationship of logarithm of multinomial probabilities to sequence entropy (circles) and relative entropy (squares).

Recently, Dekker et al. suggested that SFEs are merely a measure of sequence (Shannon) entropy (H i ), which implicitly measures how a distribution varies from equal usage [22]. (This is because the maximum entropy arises from a distribution with equal usage.)

Here, p x is the proportion of sequences with amino acid x at position i. While it is true that ΔG stat is correlated with H i (Figure 2b), this correlation is a consequence of the reference state of ΔG stat calculations being fairly close to equal usage of amino acids. It is also affected by the fact that ΔG stat values are not based on a rigorous measure of overall probability.

Plotting the multinomial probabilities associated with the distributions at the 34 positions in TPRs against sequence entropy and relative entropy clearly demonstrates that ln P mult measures the same thing as relative entropy, but something different from sequence entropy (Figure 2c). (The difference becomes more dramatic the more the reference state deviates from equal usage.) In fact, relative entropy D(p||f) is an information theory approach to measuring the "distance" between distributions, given by,

It can be shown, using the Stirling approximation for factorials, that multinomial probability is in fact linearly related to relative entropy by the number of sequences (see Supplemental Material).

One significant advantage of relative entropy over multinomial probability and ΔG stat is that relative entropy is independent of the total number of sequences N. Since the intention of the SFE approach is to estimate the significance associated with an amino acid distribution relative to a reference state, we submit that relative entropies are the most convenient way to do this. Relative entropies combine the sample-size independence and ease of calculation of Shannon entropies with the reference-state correction of Lockess & Ranganathan's method, while at the same time measuring that correction in a mathematically-rigorous way.

Other methods, in addition to Shannon entropy and the Lockless & Ranaganthan method, have been suggested for scoring residue conservation, including metrics that account for residue properties such as size or hydrophobicity [23]. There is evidence that binding sites have unique compositional preferences, which may suggest alternative reference states for our method [24]. It will be interesting to examine how attention to property variation may improve our method of dissecting binding sites in structural families.

Effects of sample size

In order to determine how many sequences are required to identify binding residues in TPRs by this method, subsets of various sizes were randomly selected from the 6,887 TPR sequences. In Figure 3a, the average relative entropy values from subsets (5 each) with approximately 6887, 3444, 1722, 861, 430, 215 and 108 randomly-selected sequences are shown for all positions. The overall pattern is evident with as few as about 100 sequences, and there is virtually no difference between subsets with 400 or more sequences. Values from the five random subsets of each size are shown for the seven lowest relative entropy positions in the full data set (Figure 3b). Again, there is essentially no discernable difference down to as few as 400 sequences, and there is not significant variability until one examines fewer than 200 sequences. We therefore expect that this analysis is applicable to protein families with as few as about 200–400 sequences.
Figure 3

Effects of sample size. (a) Average relative entropy associated with each of the 34 positions in TPRs with random subsets of various sizes. Each cluster of bars represents one position in the TPR motif. The cluster is composed of bars, left to right, from sets with approximately 6887, 3444, 1722, 861, 430, 215 and 108 sequences. Each bar is the average of five subsets of the same size (except 6887, since there is only one set this size – all sequences). (b) Relative entropies associated with five randomly chosen subsets of various sizes for the seven positions most like the reference state. Each cluster of bars represents one position. The individual bars show the calculated relative entropies for subsets of the same sizes as in (a) (five of each size).

Ankyrin repeats: comparison with experiment

A corresponding analysis of ankyrin (Ank) repeats, another experimentally well-characterized protein-binding motif, clearly confirms our prediction that low relative entropies can be used to identify specificity-determining residues when the repertoire of ligand is highly variable. Anks are helix-turn-helix-loop motifs, which bind their ligands with residues in the loops and on the surface of the helical array proximal to the loops [25]. Figure 4 shows the relative entropies from over 15,000 Ank repeats mapped onto the co-crystal structure of mouse GA binding protein β1 with the GABPα ligand bound [26]. Again, our analysis dramatically reveals that the residues known to form the binding site are among the most variable; the positions most like the reference state (relative entropy ≤ 0.39) are 2, 3, 5, 13, 14, 17, 32, and 33.
Figure 4

Relative entropy analysis of canonical positions in 15,497 Ank repeats. (a) The positional relative entropies are shown with secondary structural elements noted (grey arrows are β-strands). Blue and green arrows indicate the most variable positions; asterisks (*) indicate positions mutated by the Plückthun lab to alter Ank-domain specificity. (b) The location of the binding site in a single Ank repeat in the loop and proximal α-helical surface is labeled. (c) The 4-Ank domain from GABPβ1 (spheres) is shown bound to the ligand GABPα (ribbons) in two views from 180° rotation. Again, the binding surface is evident from the low relative entropies. Note that some non-binding surface-exposed positions, particularly turn residues, are conserved due to their importance in defining the Ank fold. Some positions in GABPβ1 do not map onto the canonical Ank sequence and are colored grey. Rendered using PyMOL from PDB entry 1AWC.

Significantly, the functional importance of the residues we have identified has already been verified experimentally. Plückthun and colleagues considered four Ank-ligand co-crystal structures and chose key interacting residues in Ank repeats from those whose solvent accessibility was most changed upon ligand binding: positions 2, 3, 5, 13, 14, and 33. Ank repeat domains which bind to different ligands, for example maltose binding protein and two kinases, have been selected from a library of Ank repeat domains in which only these positions were randomized [27, 28]. This result confirms our proposal that these are the specificity-determining residues. In fact, the crystal structure of one of the selected ankyrin proteins that binds MBP directly demonstrates the role of these residues in binding. In addition, position 32 is close in space to these residues, and may well participate in binding for some Ank proteins. It is not clear why position 17, which lies in the turn between the motif's helices, is more variable than other non-binding surface-exposed positions.

Dissecting binding sites when ligands have conserved features

We also analyzed Cys2His2 zinc fingers (Zifs) in the same way. In contrast to TPR and Ank repeats, which are stabilized by burial of hydrophobic residues, Zifs are mostly stabilized by ligation of zinc(II) and binding to DNA. Not surprisingly, then, positions that ligate the zinc ion (Cys-10, Cys-7, His+7, His+11) and a subset of positions that contact the DNA (e.g., Tyr-12, Lys-5, Phe-3, Arg+9) are highly biased (where -1 is the position immediately before the α-helix and the consensus residue is listed). Residues with low relative entropy (≤0.5) are essentially in two patches, near the end of the end of the helix buried in the major groove and on the solvent-exposed surface distal to the DNA. When one considers only the positions that are in contact with the ligand DNA, the residues with the lowest relative entropies (blue and green spheres in Figure 5) are positions -2, -1, 1, 2, 3, 5 and 6. Extensive phage-display selection work has shown that positions -1, 1, 2, 3, 5 and 6 are critical to specificity for the target DNA sequences studied [29, 30]. In contrast, positions that contact the DNA but have no effect on specificity, such as basic residues that make contacts to the phosphate backbone, are essentially invariant (orange and red spheres in Figure 5).
Figure 5

Relative entropy analysis of canonical positions in 28,442 C2H2 zinc fingers. (a) Positions in the graph are shown in the order found in Pfam and numbered by convention (where -1 is the residue N-terminal to the α-helix). Note that the y-axis scale is different from Figs. 1 and 2 due to the almost invariant zinc(II)-binding residues (-10, -7, +7 and +11). Blue and green arrows indicate the seven predicted specificity-determining positions. (b) The middle zinc finger of Zif268 bound to DNA (purple) is shown, with the Zn(II) atom as a pink sphere [[43]. (c) The residues in contact with the DNA from all three zinc fingers of Zif268 are rendered in spheres. The DNA-binding positions group into variable, specificity-determining positions (blue and green spheres) projecting into the major groove of the DNA, and conserved positions that enhance affinity to DNA but do not affect specificity (orange and red spheres). Rendered with PyMOL from PDB entry 1A1I.

Note that in this case the specificity-determining residues are not necessarily the most variable residues in the motif; they are the most variable residues in the motif that are in contact with the ligand. Thus, for Zifs, sequence hypervariation is not sufficient to identify the binding site, but statistical analysis together with a sample structure reveals specificity-determining positions without further characterization. Apparently, the repertoire of amino acids needed to bind DNA in the major groove is less diverse than that needed for the range of binding functions exhibited by TPR or Ank repeat domains.

A similar phenomenon is observed for PDZ domains, whose peptide ligands have highly conserved elements. PDZ domains are ubiquitous globular protein-protein interaction domains. It is thought that most PDZ domains bind to the C-terminus of target proteins, typically making contact to the carboxyl-terminal four to five residues. PDZ domains can be categorized into two classes (I and II) that bind to consensus sequences X-(S/T)-X-(V/I/L)-CO2 - and X-Φ-X-Φ-CO2 -, respectively (where Φ represents a hydrophobic residue and X represents an arbitrary residue) [31]. Since our method identifies residues that vary with the repertoire of ligands (i.e., specificity-determining residues), we would expect that positions that bind to the terminal carboxylate and C-terminal hydrophobic side-chain (P0) will be highly biased (i.e., conserved); positions that bind to the alcohol or hydrophobic residue at P-2 will be biased; and positions that bind to P-1 and P-3 will be highly variable. In Figure 6b, we have highlighted the positions in the example PDZ domain 3 from PSD95 that have been identified from NMR studies and X-ray co-crystal structures to be involved in binding to the terminal four residues of the ligand (323–328, 339, 340, 342, 372, 373, 376, 379 and 380) [32]. We also included positions 318, 322, 329 and 331, which are within 5 Å of the ligand peptide (KQTSV-CO2 -) in the example structure (computed with DeepView [33]).
Figure 6

Relative entropy analysis of canonical positions in 2,751 PDZ domains. (a) Positions in the graph are shown in the order found in Pfam and with the same numbering. Only positions with greater than 50% occupancy were calculated. The eight variable binding positions are marked with arrows, and the corresponding residue number in PSD95-3 is listed. (b) The structure of PSD95-3 with its cognate ligand peptide, KQTSV [[44]. Note that atoms are missing from the ligand lysine sidechain due to lack of electron density in the X-ray data. The structurally-determined binding residues (see text) are colored, and the eight predicted specificity-determining positions are labeled with residue numbers as in (a). Rendered with PyMOL from PDB entry 1BE9.

The eight most variable residues in this group are 331, 342, 376, 373, 339, 328, 340 and 326. The most variable residue, 331 (here, a glutamate), contacts the P-4 lysine, which is variable among PDZ ligands and whose effect on specificity has not been extensively examined. Positions 326, 328, 339, 340 and 342 interact with the variable P-1 and P-3 positions. Interestingly, computational redesign of PDZ domain specificity confirms the central importance of these residues in specificity determination. Reina et al. were able to change the specificity of PSD95-3 from KQTSV to KITWV and KRTEV (retaining ligand class, since P0 and P-2 are the same) [34]. In the first case, residues 326, 339, 340, 342, 380 and 397 were mutated. In the second case, residues 326, 328, 339, 340, 342 and 397 were mutated. Note that position 397 is outside of the canonical PDZ domain, and so was not examined in our work; position 380 was mutated to improve the stability of the domain, not its binding specificity.

Positions 373 and 376 contact the P-2 position, which is either an alcohol or a hydrophobic residue depending upon class. The identity of position 372 is known to be highly correlated with the ligand class, because it is typically occupied by a polar residue (often histidine) for class I ligands and by a hydrophobic residue for class II ligands [31]. As expected, position 372 displays intermediate variability (yellow in the figure). We hypothesize that positions 373 and 376 are much more variable than 372 and 380 because they are further away from the P-2 threonine; in fact, position 373 is farther than 5 Å away. These residues are likely more important for binding class II ligands in which P-2 is hydrophobic and therefore generally larger. P0 is a hydrophobic residue in virtually every known PDZ ligand, and it invariantly presents the carboxylate terminus of the peptide. Not surprisingly, then, the residues that it contacts (322–325, 327) are highly biased. In fact, position 324 is a glycine in 97% of PDZ domains, and the turn in which it lies hosts the carboxylate.

It is worth noting that, in contrast to the other three examples presented above, this calculation was carried out on a whole domain instead of a repeat motif. It is also worth noting that the binding site, as in the case of zinc fingers, could not be easily predicted from relative variability of sequence alone due to commonalities among the ligands that result in conserved elements of the binding sites. However, in combination with an example co-crystal structure, the specificity-determining positions can again be inferred from sequence variation, and the inference matches closely what has been derived from extensive biochemical characterization and engineering.

The meaning of relative entropy values

In our previous study of TPRs by SFE analysis, we empirically demonstrated that for a particular sample size and scaling, levels of sequence variation (ΔG stat ) could be usefully grouped as such: 0–1.25, hypervariable or no bias; 1.25–2.5, slight bias; 2.5–5.0, significant bias; 5.0–10.0, dramatic bias; 10+, restriction to a small subset of amino acids [16]. Regression analysis between SFE and relative entropy values for all TPR positions suggests that these values correspond to relative entropies of approximately 0.3, 0.5, 0.9 and 1.5. For convenience, we therefore chose 0.0–0.3, 0.3–0.6, 0.6–0.9, 0.9–1.2 and 1.2+ as bins for coloration of the figures in this publication. The examples in this study suggest that "normal" surface positions typically exhibit relative entropies in the range of 0.3–0.6, and that specificity-determining positions typically have relative entropies less than 0.5. The overlap of these values highlights the difficulty in using this approach as a purely predictive algorithm: only when the repertoire of ligands is extremely diverse (as with TPRs and ankyrin repeats) is there a clear distinction between ligand-binding residues and surface residues in general. We are in the process of a much broader application of this procedure to all of the families in the Pfam database, which we will use to refine the meaning of the relative entropy values in protein families overall (M. Gerstein, T. Gianoulis, T.J.M. and L.R., unpublished work).

The notion that positions that bind diverse ligands will be variable among a family of proteins seems fairly obvious, but this approach has not yet been utilized as a general strategy. One notable precedent is seen in the original studies of antigen-binding sites in antibodies, which were identified as variable regions when the amino acid sequences of antibodies were first determined [35]. Various family-based approaches have been applied to the prediction of functional residues, typically analyzing sequence variability from collections of proteins with similar function (and therefore emphasizing the functional importance of conserved residues). For example, "evolutionary trace" and related methods divide multiply-aligned sequences into subfamilies, typically by phylogeny, comparing patterns of conservation among evolutionarily-related subfamilies and often mapping onto 3D structure [13, 3642]. Basically, these methods posit that positions that are conserved in all sequences are important for structure, and positions that are conserved within subfamilies (but vary among the sub-types) are important for function (i.e., the function of the proteins in the subfamily).

Here we show how analysis of sequence variability can be enlarged to understand functional variability in whole families of proteins with similar structures. If one collects proteins with the same structure and diverse functions, then structural positions will be conserved and functional positions will vary, and the degree of variation will be related to the degree of variation among the ligands or substrates. In the case of repeat motifs such as TPRs and ankyrins, where structural elements are further divorced from functional conservation by ignoring how the motifs are arranged in domains, the degree of variability is sometimes so profound that it alone can be used to predict the binding site. When the ligands have commonalities, then it becomes more difficult to predict the binding site from variation alone. In that case, as with PDZ domains and Zifs, variation in combination with an example structure still reveals specificity-determining binding positions, which is critical information for re-engineering specificity. (The corollary to this argument is that the pattern of variation of known binding residues will suggest the pattern of variation in the ligands.)

Analysis of overall variance among structurally-related families provides complementary information to methods that analyze variance among evolutionarily-related subfamilies, which have proven very powerful in recent years. A major challenge for these evolutionary trace methods is accurate functional sub-typing, particularly when family members have diverged very significantly. Our method avoids functional sub-typing and, rather, benefits from increased functional divergence of family members (since it results in increased variation among functional positions). Further attention to variable residues in families overall therefore stands to improve exiting methods of functional prediction.

There are hundreds of binding scaffolds with sufficient examples known to permit this type of statistical analysis. The use of rigorous measures of how amino acid distributions differ improves significantly upon conservation alone as a means of identifying important residues within a protein family (this has been reviewed recently [23]). The rapid identification of specificity-determining positions will be useful for the design of proteins with altered binding specificity. The predictions of specificity-determining residues in Ank repeat proteins, Zifs and PDZ domains agree strikingly well with results from considerable structural and biochemical work, and therefore provide a guide for re-engineering binding specificity by design even for protein families lacking extensive characterization. Moreover, knowledge of the specificity-determining residues can be incorporated into evolutionary trace methods to develop a comprehensive view of residues critical for function.

Aligned sequences of TPRs, Ank repeats, C2H2 Zifs and PDZ domains were downloaded from Pfam http://pfam.wustl.edu[18]. TPRs of non-canonical length (i.e., not 34 amino acids) were discarded, and only canonical positions were considered with Ank repeats, Zifs and PDZ domains (i.e., ignoring low-occupancy positions from insertions and deletions). All calculations were carried out in Microsoft Excel 2003 on a Dell Latitude C640 with a 2.2 GHz Intel Mobile Pentium 4 processor. Factorials were computed from the Stirling approximation.

Statistical Free Energies

The SFEs associated with each amino acid were determined from application of the Boltzmann law [1], where k is the Boltzmann constant, kT* is an arbitrary energy unit (since the "temperature" of the ensemble T* is not necessarily related to T for conventional systems), and P ref is the probability associated with a hypothetical site with amino acid usage as in the reference state. The binomial probability P x of the observation of n x sequences with amino acid x was calculated from [3], where f x is the frequency of the amino acid in a reference state, all ORFs in Saccharomyces cerevisiae. The SFEs associated with the observed frequencies of the 20 amino acids at each site can be thought of as elements of a 20-dimensional vector. The scalar length of this vector, the root-mean-square average for all amino acids [2], is therefore taken to be the statistical free energy, ΔG stat , that separates the observed positional amino acid distribution from the reference state. For comparison to Lockless & Ranganathan [14], the ΔG stat values were arbitrarily divided by 100. However, the Ranganathan group normalizes the number of sequences to 100, and we have shown that ΔG stat is proportional to N for large N. Therefore, the ΔG stat values we calculate are 10-fold larger than those calculated by the Ranganathan method for the same amino acid distribution, since we have normalized to N = 1000.

Other metrics of distribution difference

Multinomial probabilities [4], sequence (Shannon) entropies [5] and relative entropies [6] were calculated as described above. For sequence and relative entropy calculations, the frequencies were calculated as:

so that ln p x was always defined (and is valid as long as N is large). The values used for f x are listed in our previous paper [16].

List of abbreviations

Hsp-organizing protein

maltose binding protein

statistical free energy

tetratricopeptide repeat protein

zinc finger

We thank A. López Cortajarena, T. Kajander, S. Mochrie, J. Venkatraman and C.G.M. Wilson (Yale) and S.S. Licht (MIT) for critical reading of this manuscript and insightful comments. Special thanks to M. Gerstein (Yale) for helpful suggestions on calculating distribution differences. T.J.M. was an N.I.H. Postdoctoral Fellow (GM065750). This work was supported in part by N.I.H. grants GM49146 and GM62413 (L.R.).

Authors’ Affiliations

Department of Molecular Biophysics & Biochemistry, Yale University
Present address: Department of Chemistry and Department of Biochemistry, The Ohio State University
Department of Chemistry, Yale University

  1. Teichmann SA, Murzin AG, Chothia C: Determination of protein function, evolution and interactions by structural genomics. Current Opinion in Structural Biology 2001, 11:354–363.View ArticlePubMed
  2. Thornton JM: From genome to function. Science 2001, 292:2095–2097.View ArticlePubMed
  3. Gough J, Karplus K, Hughey R, Chothia C: Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. Journal of Molecular Biology 2001, 313:903–919.View ArticlePubMed
  4. Bork P, Koonin EV: Protein sequence motifs. Current Opinion in Structural Biology 1996, 6:366–376.View ArticlePubMed
  5. Whisstock JC, Lesk AM: Prediction of protein function from protein sequence and structure. Quarterly Review of Biophysics 2003, 36:307–340.View Article
  6. Yao H, Kristensen DM, Mihalek I, Sowa ME, Shaw C, Kimmel M, Kavraki L, Lichtarge O: An accurate, sensitive, and scalable method to identify functional sites in protein structures. Journal of Molecular Biology 2003, 326:255–261.View ArticlePubMed
  7. Ma B, Elkayam T, Wolfson H, Nussinov R: Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces. Proceedings of the National Academy of Sciences of the United States of America 2003, 100:5772–5777.View ArticlePubMed
  8. Jones S, Thornton JM: Searching for functional sites in protein structures. Current Opinion in Chemical Biology 2004, 8:3–7.View ArticlePubMed
  9. Mihalek I, Res I, Lichtarge O: A family of evolution-entropy hybrid methods for ranking protein residues by importance. Journal of Molecular Biology 2004, 336:1265–1282.View ArticlePubMed
  10. Chothia C, Lesk AM: The relation between the divergence of sequence and structure in proteins. EMBO Journal 1986, 5:823–826.PubMed
  11. Bashford D, Chothia C, Lesk AM: Determinants of a protein fold. Unique features of the globin amino acid sequences. Journal of Molecular Biology 1987, 196:199–216.View ArticlePubMed
  12. Bowie JU, Reidhaar-Olson JF, Lim WA, Sauer RT: Deciphering the message in protein sequences: tolerance to amino acid substitutions. Science 1990, 247:1306–1310.View ArticlePubMed
  13. Lichtarge O, Bourne HR, Cohen FE: An evolutionary trace method defines binding surfaces common to protein families. Journal of Molecular Biology 1996, 257:342–358.View ArticlePubMed
  14. Lockless SW, Ranganathan R: Evolutionarily conserved pathways of energetic connectivity in protein families. Science 1999, 286:295–299.View ArticlePubMed
  15. Shortle D: Propensities, probabilities and the Boltzmann hypothesis. Protein Science 2003, 12:1298–1302.View ArticlePubMed
  16. Magliery TJ, Regan L: Beyond consensus: statistical free energies reveal hidden interactions in the design of a TPR motif. J Mol Biol 2004, 343:731–745.View ArticlePubMed
  17. D'Andrea LD, Regan L: TPR proteins: the versatile helix. Trends in Biochemical Sciences 2003, 28:655–662.View ArticlePubMed
  18. Sonnhammer EL, Eddy SR, Durbin R: Pfam: a comprehensive database of protein domain families based on seed alignments. Proteins 1997, 28:405–420.View ArticlePubMed
  19. Scheufler C, Brinker A, Bourenkov G, Pegoraro S, Moroder L, Bartunik H, Hartl FU, Moarefi I: Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 2000, 101:199–210.View ArticlePubMed
  20. Gatto GJJ, Geisbrecht BV, Gould SJ, Berg JM: Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nature Structural Biology 2000, 7:1091–1095.View ArticlePubMed
  21. Kanehisa M, Goto S, Kawashima S, Nakaya A: The KEGG databases at GenomeNet. Nucleic Acids Research 2002, 30:42–46.View ArticlePubMed
  22. Dekker JP, Fodor A, Aldrich RW, Yellen G: A perturbation-based method for calculating explicit likelihood of evolutionary co-variance in multiple sequence alignments. Bioinformatics 2004, 20:1565–1572.View ArticlePubMed
  23. Valdar WS: Scoring residue conservation. Proteins 2002, 48:227–241.View ArticlePubMed
  24. Villar HO, Kauvar LM: Amino acid preferences at protein binding sites. FEBS Lett 1994, 349:125–130.View ArticlePubMed
  25. Sedgwick SG, Smerdon SJ: The ankyrin repeat: a diversity of interactions on a common structural framework. Trends in Biochemical Sciences 1999, 24:311–316.View ArticlePubMed
  26. Batchelor AH, Piper DE, de la Brousse FC, McKnight SL, Wolberger C: The structure of GABPalpha/beta: an ETS domain- ankyrin repeat heterodimer bound to DNA. Science 1998, 279:1037–1041.View ArticlePubMed
  27. Binz HK, Amstutz P, Kohl A, Stumpp MT, CBriand C, Forrer P, Grutter MG, Pluckthun A: High-affinity binders selected from designed ankyrin repeat protein libraries. Nature Biotechnology 2004, 22:575–582.View ArticlePubMed
  28. Binz HK, Stumpp MT, Forrer P, Amstutz P, Pluckthun A: Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. Journal of Molecular Biology 2003, 332:489–503.View ArticlePubMed
  29. Wolfe SA, Nekludova L, Pabo CO: DNA recognition by Cys(2)His(2) zinc finger proteins. Annual Review of Biophysics & Biomolecular Structure 2000, 29:183–212.View Article
  30. Pabo CO, Peisach E, Grant RA: Design and selection of novel Cys(2)His(2) zinc finger proteins. Annual Review of Biochemistry 2001, 70:313–340.View ArticlePubMed
  31. Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC: Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 1997, 275:73–77.View ArticlePubMed
  32. Wiedemann U, Boisguerin P, Leben R, Leitner D, Krause G, Moelling K, Volkmer-Engert R, Oschkinat H: Quantification of PDZ domain specificity, prediction of ligand affinity and rational design of super-binding peptides. J Mol Biol 2004, 343:703–718.View ArticlePubMed
  33. Guex N, Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 1997, 18:2714–2723.View ArticlePubMed
  34. Reina J, Lacroix E, Hobson SD, Fernandez-Ballester G, Rybin V, Schwab MS, Serrano L, Gonzalez C: Computer-aided design of a PDZ domain to recognize new target sequences. Nat Struct Biol 2002, 9:621–627.PubMed
  35. Hilschmann N, Craig LC: Amino acid sequence with Bence-Jones proteins. Proceedings of the National Academy of Sciences of the United States of America 1965, 53:1403–1409.View ArticlePubMed
  36. Sali A, Overington JP, Johnson MS, Blundell TL: From comparisons of protein sequences and structures to protein modelling and design. Trends Biochem Sci 1990, 15:235–240.View ArticlePubMed
  37. Hannenhalli SS, Russell RB: Analysis and prediction of functional sub-types from protein sequence alignments. J Mol Biol 2000, 303:61–76.View ArticlePubMed
  38. Innis CA, Shi J, Blundell TL: Evolutionary trace analysis of TGF-beta and related growth factors: implications for site-directed mutagenesis. Protein Eng 2000, 13:839–847.View ArticlePubMed
  39. Landgraf R, Xenarios I, Eisenberg D: Three-dimensional cluster analysis identifies interfaces and functional residue clusters in proteins. J Mol Biol 2001, 307:1487–1502.View ArticlePubMed
  40. del Sol Mesa A, Pazos F, Valencia A: Automatic methods for predicting functionally important residues. J Mol Biol 2003, 326:1289–1302.View ArticlePubMed
  41. Folkertsma S, van Noort P, Van Durme J, Joosten HJ, Bettler E, Fleuren W, Oliveira L, Horn F, de Vlieg J, Vriend G: A family-based approach reveals the function of residues in the nuclear receptor ligand-binding domain. J Mol Biol 2004, 341:321–335.View ArticlePubMed
  42. Berezin C, Glaser F, Rosenberg J, Paz I, Pupko T, Fariselli P, Casadio R, Ben-Tal N: ConSeq: the identification of functionally and structurally important residues in protein sequences. Bioinformatics 2004, 20:1322–1324.View ArticlePubMed
  43. Elrod-Erickson M, Benson TE, Pabo CO: High-resolution structures of variant Zif268-DNA complexes: implications for understanding zinc finger-DNA recognition. Structure 1998, 6:451–464.View ArticlePubMed
  44. Doyle DA, Lee A, Lewis J, Kim E, Sheng M, MacKinnon R: Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 1996, 85:1067–1076.View ArticlePubMed

© Magliery and Regan. 2005


Solidarity and ‘the veil’: Why wearing a hijab in solidarity is more complicated than you think

by: Farah Azadi

Lately, there have been many stories of non-Muslim women deciding to wear the Muslim head scarf (or hijab) as a way of showing solidarity with Muslims, especially Muslim women, in this age of growing Islamophobia. One example is the Wheaton College professor, Larycia Hawkins, who is donning the hijab for the Christian season of Advent, explaining, ‘as part of my Advent Worship, I will wear the hijab to work at Wheaton College, to play in Chi-town, in the airport and on the airplane to my home state that initiated one of the first anti-Sharia laws and at church.’

There are many other examples, from Facebook groups like Women in Solidarity with Hijabis (WISH) to #40daysofhijab and the ‘hijab selfies’ movement where women, many who self-identify as ‘committed Christians’, post a picture of themselves on social media wearing the hijab ‘out of solidarity’.

Where I think it is important to acknowledge the very genuine motivations behind these acts of ’embodied solidarity’ their efficacy should also be considered.

In a climate of growing Islamophobia in Europe and the U.S., and a narrowing of the political nuances around identity, power and politics, many have attempted to consider how to engage with their Muslim neighbours, colleagues, and friends. For Muslims, as for others who experience violence, hatred or oppression based on their identity(ies) it is  encouraging to see others, outside one’s own community, acknowledge and engage with this injustice. It communicates not simply compassion, but a willingness to act, to speak up, to work to change the status quo.

At the same time, it is good to realise that solidarity, as opposed to charity, works to build relationships of mutual aid. In this way, one does not act simply on conscience, but interrogates why an inequitable relation exists in the first place. As Helder Camara, a Brazilian priest, once said during a period of great repression against the poor in Brazil, ‘Quando dou comida aos pobres chamam-me de santo. Quando pergunto por que eles são pobres chamam-me de comunista.’ (‘When I give food to the poor, they call me a saint. When I ask why they are poor, they call me a communist.’)

After nearly two decades of working at the grassroots level, with campaigns and movements organising for global equity, it is those movements that are led by the communities most affected that sustain the most meaningful change. I argue the same is true in this situation, Muslims are the ones best suited to speak to and to lead discussions about combating Islamophobia.

Of course in the case of the hijab, it is important to realise that there are almost as many opinions as there are Muslim women. Where some Muslim women wear hijab to symbolise their piety, freedom of expression and feminism; others see the hijab as a symbol of women’s subordination. Similarly, as Muslims make up nearly 22 percent of the world’s population and come from nearly every country of the world, there are a myriad of ethnic and/or national cultural traditions that come through dress, including ‘hijab style’. For instance, the burqa, a traditional hijab style from Afghanistan looks very different from styles worn in Malaysia, Lebanon or the Gulf States.

It is also important to realise that within this diversity, are contrasting attitudes towards the hijab at the state level. In Iran, for instance, following the 1979 ‘Islamic Revolution’ the hijab became mandatory for all women (or people presenting as women). Whereas, in states such as Egypt, Albania, Indonesia or Guinea, all Muslim majority states, there is no legislation regarding hijab.

This is why attitudes and experiences of hijab differ greatly within the Muslim community, and why it is important for non-Muslims to think through how their acts of solidarity have an effect on the community they are standing with. For instance, what does wearing the hijab for reasons of solidarity communicate to women whose experience with mandatory hijab is painful or humiliating? What does it communicate to Muslim women who for political, religious or feminist reasons choose not to wear hijab?

It is also important to remember that where wearing hijab might appear to be ‘adventurous’, many women’s choice to wear it is endemic to who they are and what they most fervently believe. In this way it isn’t easily abandoned, even where women may be targeted, abused or suffer violence because of it. For example, post 9/11, Islamophobia spiked and instances of racial violence against women wearing hijab made up the most significant portion of those targeted. Where some woman chose to stop wearing hijab for this reason, the majority of others continued wearing the hijab despite this violence. In fact, often when violence in the name of Islam is perpetuated (largely by men), it is Muslim women who must endure the backlash.

These are the very real, embodied experiences (alongside many very positive ones), that comprise the depth and breadth of experiences of wearing the headscarf—and they hardly scratch the surface of what it means to be a ‘Muslim woman’. For me, these examples provide insight into the complexity of this issue, and why donning the headscarf, even out of solidarity, needs to be more considered.

Further, I think that we can choose paths of solidarity without having to appropriate or become the people or things we are in solidarity with. (An important point made during the Rachel Dolezal controversy). Instead, making and/or giving space to marginalised communities to speak for themselves is often the best way to ally.

by: Gayatari Spivak

Collective hatred comes from narratives of cultural memory.

In 1916, anticipating victory, France, Russia, and Britain created the “Middle East” out of the remains of the 600-year old Ottoman Empire. Lebanon and Iraq were directly controlled, others kept in spheres of influence. Haifa, Gaza, and Jerusalem were an Allied “condominium.” Arms control was strictly European. The Arab powers learned of this at war’s end (1917). Agreements assuring Arab independence had disappeared.

Such are the ingredients for a future cultural memory.

The Ottoman Empire was corrupt but, except for focused examples such as the Armenian genocide, generally carried an attitude of conflictual co-existence toward religious difference. Now arrived a master race that thought itself justified in controlling and systematizing the locals, without any social contract, often by remote control. An inchoate resentment stirred in people at ground level who could not combat this transformation. Women felt it strongly, thinking of men as holding their dignity. The skeleton of a cultural memory in the making now fleshes out.

With the Balfour Declaration (1917), approved by the League of Nations (1922), Britain is charged to administer parts of the defunct Ottoman Empire, “until such time as they are able to stand alone.”

Nothing should be done to prejudice the civil and religious rights of existing non-Jewish communities in Palestine, the declarations say. Now the sense of a religious as well as civil right is ready to form without internal institutional intellectual support, and the narrative of “cultural memory” thickens further. The outrage is strongest in those–less privileged, landlocked–who are made to feel that they do not deserve to live on their land.

After 1948, the power that had passed from Ottoman to Europe, passes to United States and Israel. Israel begins to justify itself by cultural memory: biblical narrative. The question of the right to religion solidifies, transformed into the new abstract idiom of the state. For Israel this is sharpened by past European oppression and denial of Europeanness. In Palestine, however, the right to land as sacred space cannot invoke that pre-history as justification for the displacement of original inhabitants, who also now begin to inhabit religious rights discourse.

Islam is international. The discourse of religion permitted connections: with the CIA-backed Taliban in Afghanistan, the post-independence recoding of Hindu-Muslim conflictual coexistence upon the Indian subcontinent, the emergence of the Wahhabis, consequences of the Shah’s deposition in Iran, and, after 1989, the “Islamic” post-Soviet bloc. Cultural memory as “religion” can now create an ideology of just war through early childhood education of the deprived.

After World War II, the United States picked up Europe’s burden. And “America” seemed to get away with everything–remaining the repository of Enlightenment virtues, the shining land where immigrants flock. Yet, looking at Haiti, the Congo, or Chile–Aristide, Lumumba, Allende, the list goes on–it seems absurd to say that America stands for justice and right. And Israel is regularly described as the only democracy in the region.

That’s why “they” want to harm “us”–because, for a long time, “we” seem to have wanted to harm “them,” and own “them,” for no reason at all: imperial foreign policy, narrativized into cultural memory. Yet “we” are the angels. As a literary critic/activist/educator, I think to find such causes–though I applaud Helen Thomas’s tenacity–is as counterproductive as avoiding the question. For the point is to dislodge the polarization, unmake narrative, undo memory. Impossible tasks.

Amnesty International: Human Rights are meaningless without human solidarity.

In a speech for Amnesty International’s  International Council meeting in Dublin yesterday Michael D. Higgins called the EU response to the migrant crisis ‘shameful’:

We must acknowledge that the European response as a whole has been grossly inadequate….This failure can only be described as shameful and undermines both the ideal of the European Union and any prospect of that Union being an exemplar for international law and its instruments.

On his twitter account today Colm O’Gorman the Executive Director of Amnesty International Ireland quoted this line from Higgins’ speech in an apparent affirmation with the Presidents’ sentiments. Where there is clearly nothing to fault about such sentiments, their expression by these two well-known political figures should be fortified by an equal measure of action, and particularly moral leadership when it comes to a crisis that so visibly challenges the universality of the concept of human rights.  However, the words of Higgins and their echo by O’Gorman are nothing more than pretence when not forged within meaningful commitments to action or to support individuals whose lives are gripped by such pernicious failures.

Amnesty International began in 1961 after an article about two Portuguese students imprisoned for holding a ‘toast to freedom’ sparked a British journalist to author a piece which inspired the ‘Appeal for Amnesty 1961,’ launching an international movement highlighting the plight of prisoners of conscience. That movement today is Amnesty International (AI), one of the most well-known human rights organisations in the world.

Since its founding AI’s work has evolved in many directions, yet there has always been a consistent core focus on incarceration especially for those imprisoned ‘in defence of freedom of opinion and religion.’ The focus of AI on prisoners of conscience has raised awareness worldwide about the right to freedom of speech, and more generally over the importance of narrative in the shaping of political events and consciousness.

This is precisely the reason that Higgins’ speech is in some ways exceptionally critical at this juncture, where there is a pressing and crucial need to challenge the rhetoric of European leaders who would use the migrant crisis to further deflect from the growing indignities resulting from austerity and its grounding in neoliberal capitalism.

In contrast to the racist conclusions that groups such as Identity Ireland peddle in their misguided attempt at political populism, an appeal to ‘human rights’ and commitment to ‘anti-racism’ cannot be faulted—everyone from Fine Gael to the Socialist Party can agree that racism is ‘wrong’ and that human rights are ‘good’. Yet it is precisely what those values mean in practice that produces their most consequential significance as concepts we have come to see as so fundamental to our world-view/s, and thus so important to examine.

Which is why I feel it is critical to challenge both President Higgins and AI – Ireland in drawing the perceptible lines of connection between a commitment to human rights in word and its exercise in deed. To me this means where Michael D. Higgins calls the ‘migrant crisis’ shameful I do wonder why there is a studied silence as to how those migrants are treated after they have been ‘rescued’? Does their being locked away in detention centres or concentrated into camps such as those in Calais or across Greece or Italy with no recourse to social protections not also shame us?

A few weeks ago a letter went out to the Department of Justice calling for the immediate release of an Afghan refugee found in Naas. Many will know what subsequently happened to him being imprisoned on the criminal charge of not having documents, and then subsequently brought three times before the court on the same charge only for the reason that the person responsible for his welfare was incompetent and refused to file the paperwork that would see his immediate release.

The letter warned against the dangerous precedent of further criminalising migration and compounding trauma through treating refugees in this manner, and while multiple NGOs who advocate for migrants, refugees and human rights signed, AI – Ireland remained silent.

This failure provokes questions over how an organisation so dedicated to the issues of imprisonment, protection and human rights can so thoroughly fail at connecting those values on which they claim their organisation’s founding principles and ongoing legitimacy. As Angela Davis has said, ‘prisons do not disappear problems, they disappear people,’ and claiming the prison system is an adequate place to address the myriad of issues presented by an undocumented, traumatised and extremely vulnerable Afghan refugee not only shows a lack of understanding of contemporary migration, it reveals a poverty of compassion.

Amnesty claims that its work today has grown

from seeking the release of political prisoners to upholding the whole spectrum of human rights. Our work protects and empowers people…from combatting discrimination to defending refugees and migrants’ rights. We speak out for anyone and everyone whose freedom and dignity are under threat.

I hope that AI – Ireland takes the opportunity afforded by the attention shone on its work through Higgins’ remarkable speech to nuance its understanding of contemporary migration and to speak out against the many forms of imprisonment, from direct provision to the criminalisation of the undocumented, that present contemporary examples of ‘prisoners of conscience’ upon which their movement moves.

by: Farah Mokhtareizadeh

Walli Ullah Safi, 21 years of age, has been in Cloverhill prison for more than two weeks.

In very different circumstances, I was in prison at the same age for two weeks after protesting the ongoing wars in Iraq and Afghanistan. It was one of hundreds of demonstrations I had been on after returning to the United States from Iraq. I had first travelled to Iraq, just barely 18 years of age, with a campaigning group who had highlighted the humanitarian crisis Iraqis faced under the sanctions. In the aftermath of the 9/11 attacks, the group had rallied in opposition to the Iraq war, mobilising thousands onto the streets.

Those years protesting the Iraq war were the most formative political experiences of my life, but being in prison had a haunting, and honestly chilling effect on my activism. I became all too aware of the complex matrix of issues that resulted in people engaging in ‘illegal’ action, and soon felt rather ashamed at the fact that I had consciously made the decision to go to prison when so many of the women who shared my experience were largely forced into the prison system due to their lack of privilege, knowledge of their rights or simply the absence of a way to make those rights actionable.

Walli Ullah Safi is a young man from Afghanistan and that is as much as I can confirm that I know about him. However, unlike those given the responsibility over his care, I do know more generally about the context and circumstances that may have affected his decision to leave Afghanistan. Though I have never travelled to the south of Afghanistan where the war between the international forces, including Ireland, and those of the opposition has never really ended, I have met many people who had to flee the south due to drone bombings, night raids, paramilitary incursions against ‘occupation friendly’ villages, military incarcerations, torture, rape—including that of children, and any myriad of brutalities that have resulted directly from decades upon decades of serial war. Walli, based on his language being Pashto, is very likely native to the south of Afghanistan where war has characterised most of his life—a prison of another form.

But war isn’t inherent to Afghan society, despite what the ‘graveyard of empires’ narrative would suggest. Rather, war has been visited upon Afghanistan time and again by the remorseless calculations of empire, beginning with the British occupations in the 1840s. At the start of the potato famine and mass emigration in Ireland, where the British deprived its starving colonial subjects the most basic necessities of human substance, they spent their fortune spilling the blood of Afghans in an attempt to constrain the growing dominance of another imperial state—Russia. Since that time, Afghanistan has been used by different colonial powers (the British, the Soviets, the Saudis and the Americans) to service ambitions I can only describe as frivolities.

The common experiences of colonial occupation and oppression should, for anyone with a sense of history, present a tangible and direct connection to the Afghans despite the myriad of differences of culture, language, religious background and colour between ‘us’ and ‘them’. This perceptible link should be the context in which those so-called representatives of the Irish people lead this island in moral and political courage, fortified by the sagacity, savage as it is, of our own past and renewed by the remarkable commitment to equality demonstrated earlier this year.

Walli Ullah Safi, if like the other young people I met across Afghanistan, has experienced more violence than most could imagine in a lifetime. In contrast with the chauvinistic rhetoric of the American government, the ongoing violence that condemns Afghans largely to a lottery of poverty or death will inevitably lead the determined to resist or to migrate. That migration is about survival, a defiance of the American attempt to ‘carve 9/11 into the history of loss in other places.’

In many ways, Walli Ullah Safi’s experiences acts as a mirror, the looking glass through which we can regain our sense of history. These bare facts and what can be extrapolated from them about Walli Ullah Safi and the circumstances of his life echo the conditions that circumscribe the lives of millions of people worldwide. In this, Walli Ullah Safi is representative of the punitive existence that faces those imprisoned in a myriad of realities in which their agency is deprived of its actionability.

His plight is also a reflection of the callousness of the Irish state and the European Union whose stance concerning the misery of the Greeks is incomparable in many ways to those whose hue, religious proclivities and circumstance render them as less than human ‘cockroaches’, a ‘swarm’ in Mr Cameron’s estimation, whose occupying troops only left Afghanistan this week after 14 years.

What troubles me most, however, is that Walli Ullah Safi is also an augur. His story typifies what the future will hold for these ‘undesirables’ we label ‘migrant’, ‘asylum seeker’, ‘refugee’, ‘illegal’…but no amount of legal terminology can cloak the flesh and blood young person suffering needlessly in Cloverhill prison.

He doesn’t need to be there, the law ironically is on his side—the section that holds him does not apply to refugees or to people who have made an application for asylum, as we are told Walli Ullah Safi has done. If Walli’s best interest were foremost, the demand for an immediate application for habeas corpus under article 40.4.2 of the Irish constitution would be called for by every migrant rights organisation following this case. The questions loom as to why these have not been done, and a traumatised young man seeking protection remains in a place where, after last week’s events, he is not safe.

Yet Walli Ullah Safi remains in prison due to a mix of incompetence and an aversion to accountability that so characterises the many institutions of the Irish state.The intrepid prophesier himself, James Baldwin, imparted that ‘the relationship of morality and power is a very subtle one…because ultimately power without morality is no longer power.’ What is it that threatens so the imperium of the Irish state and its E.U. collaborators about cockroaches? One day, maybe soon, they may inherit the earth.

french algeria

Talk Notes: After Charlie Hebdo: complex media cultures and the limits of liberalism

Impossible Solidarities: Islam, Feminism and (fortress) Europe’s shifting frontiers

Whilst co-organising a vigil this past week commemorating those who drowned in the Mediterranean attempting to breach fortress Europe I came across a passage by the late James Baldwin that I haven’t been able to stop thinking about. The passage comes from an interview Baldwin did for the NY Times on the occasion of his 52nd birthday and his return to New York after more than a decade in Paris. Time constraints wont’ allow me to give you much of a background, but given the ways in which racism and the tropes it perpetuates again and again remain as prevalent today as they were in 1977 when this article was published its perhaps better you hear it as if Baldwin is speaking about Baltimore or Ferguson or even Lampadusa or Calais when he says,

there is a history w all have to contend with…For a long while, liberty was a privilege in this country–if you’re doing well, you can shout to your heart’s content, provided no one starts listening to you and your message doesn’t threaten too many people. We act as if this is a free country, until the White people tell us its not by jailing us or killing us. And a lot of us have been locked up or murdered over the centuries we’ve been here. Its a hard thing to talk about…Some people have tears in their eyes and let me know how awful they feel about the way our poor live, our blacks, or those in dozens of other countries, but people can cry much easier than they can change.

Continue reading

women with stash

Talk Notes from Kieran Flynn Memorial Lecture Series on Islam in the West

The silence around feminism and religion is a profound one, and I think some of its roots lie in the narrative of secularism and its influence on feminism in both the academy and in feminist social movements. I think the silence functions to highlight a difficulty in approaching the subject of female autonomy in relation to religion, but also indicates a negativity towards religion on the part of feminist scholars –justified or not.

Although there has been a significant amount of work on religion and patriarchy (Dominance of a society by men, or the values that uphold such dominance.) as well as on agency, autonomy, and gender; there has less on the subject of women, religion and autonomy. Continue reading

(Un)making Idolatry From Mecca to Bamiyan

By: Jamal Elias

Note: A pdf of this article, inclusive of pictures and captions that we have not been able to reproduce in this format, can be found here

I ask the Afghans and the Muslims of the world: Would you rather be the smashers of idols or the sellers of idols? – Mullah Umar, supreme leader of the Taliban

It is not those who forget, but those who “remember” the past that are condemned to repeat it. -Sheldon Pollock, “Ramayana and Political Imagination in India” Continue reading

The Charlie Hebdo killers were operating under a misapprehension. TOPKAPI PALACE LIBRARY

In the wake of the massacre that took place in the Paris offices of Charlie Hebdo, I have been called upon as a scholar specializing in Islamic paintings of the Prophet to explain whether images of Muhammad are banned in Islam.

The short and simple answer is no. The Koran does not prohibit figural imagery. Rather, it castigates the worship of idols, which are understood as concrete embodiments of the polytheistic beliefs that Islam supplanted when it emerged as a purely monotheistic faith in the Arabian Peninsula during the seventh century.

Continue reading

Get every new post delivered to your Inbox.

Join 663 other followers


Web Results
Legal Term Stayed | Define Legal Term Stayed at Dictionary.com
dictionary.reference.com/browse/Legal Term Stayed

Stay of proceedings

A stay of proceedings is a ruling by the court in civil and criminal procedure, halting further legal process in a trial. The court can subsequently lift the stay and  ...

Stay legal definition of stay

Stay. The act of temporarily stopping a judicial proceeding through the order of a court. A stay is a suspension of a case or a suspension of a particular ...

Stay Definition - Duhaime.org

The legal definition of Stay is To stop; to suspend; also known as a stay of proceedings; when a law suit is suspended either indefinitely or until the occurrence of ...

stay - Legal Dictionary | Law.com

stay. n. a court-ordered short-term delay in judicial proceedings to give a losing defendant time to arrange for payment of the judgment or move out of the ...

Stay Law & Legal Definition

What does the legal term "stayed and imposed" mean? - Ask.com

"Stayed and imposed" means that a criminal court judge imposes a sentence but stays that sentence, meaning that the convict does not immediately serve the ...

Stay of Proceedings | Wex Legal Dictionary / Encyclopedia | LII ...

A court may later lift the stay and continue the proceeding. ... Usually, the pendency of an appeal usually stays proceedings in the court below. In Long v.

Stay Law | Definition of Stay law by Merriam-Webster

www.merriam-webster.com/dictionary/stay law

a moratory law : a law suspending or providing a means of suspending execution of judgments or sale on foreclosure or otherwise suspending legal remedies ...

What is stay order? definition and meaning - BusinessDictionary.com

Definition of stay order: Court order that suspends a judicial proceeding (or a judgment resulting from ... Popular 'Corporate, Commercial, & General Law' Terms.

Popular Q&A
Q: What does the legal term stay mean.
A: Typically the word stay means to put on hold, such as a stay in execution. It is a temporary pause of proceedings usually used to determine extent of evidence. ... Read More »
Source: www.answers.com
Q: What does the legal term stayed mean.
A: Stayed in legal terms means the act of temporarily stopping a judicial proceeding through the order of a court.! Read More »
Source: www.chacha.com
Q: What does the legal term "Stay" mean?
A: To hold back or not immediately enforce, like a stay of execution Read More »
Source: answers.yahoo.com
Q: What does the legal term relief from stay mean?
A: Relief from stay is a request by a creditor to permit the creditor to enforce its claims and pursue collection activity. ! Read More »
Source: www.chacha.com
Q: What is a stay in legal terms.
A: A stay occurs after a judge issues a ruling. Now let's say the ruling tells person A they cannot be within 50 feet of person B. A stay issued either by the disc... Read More »
Source: www.answers.com


ENB:05:73 [Next] . [Previous] . [Contents]

NGOs are reportedly rallying in reaction to amendments circulated by the OPEC States to the Co-Chairs’ text on energy, transport and atmosphere. One NGO representative said the proposed amendments delete language stressing the importance of adopting a protocol or another legal instrument at FCCC COP-3 and add references to the inability to meet current commitments on lowering emissions and the adverse impacts on economic development. It also modifies language on increased investment in renewable energy research and the damaging impacts of fossil energy use by transportation systems.


Language ≡ Sections

Topic Teasers Vol. 70: Compliance Challenges

Question: We need to make some alterations in the way we manage our internal software and the access employees have to protected data. Now an audit shows we need to make specific changes immediately or pay a huge fine. Is there any way to avoid this embarrassment in the future and protect our public image?
A. International and federal regulations must be complied with, and knowing which regulations apply to your type of business so you can merge compliance into your plans at the project level will help satisfy the mandatory requirements to avoid large fines in the future.
B. Most staff members are reluctant to move forward with required changes to their daily routine, whether technological or manual. To get their cooperation and meet the audit list, fire the first three people who speak out in opposition. Setting an example clearly conveys that this change will occur with or without the agreement of the employees.
C. If your organization has been running smoothly and profitably, ask the legal team to search the past rulings to find ways to avoid altering the violation items listed by the compliance inspectors.
D. Each type of business has a single federal mandate to govern how they manage their profits. Know that the depreciation versus capitalization of corporate expenses should be recorded and tracked to meet audit specifications.
Pick your answer then Test Your Knowledge!

Slashing Risks with User Experience Engineering (Part 4)

by Dmitri Khanine

This is the fourth and final installment in this series on using the latest UX methods for focusing on the right problems and slashing requirements-based risks. In this installment, we will be validating designs, using our prototypes for conducting usability tests.

Slashing Risks with User Experience Engineering (Part 3)

by Dmitri Khanine

What is UX, and why should you pay attention? In the first article, we looked at the seven key UX activities involved in collecting accurate insights, modelling and validating our designs. Part 2 focused exclusively on the key differences of modern user research methods from traditional requirements-gathering activities. Now we look at building prototypes that will make it easy for us to later validate our solutions with usability testing.

Slashing Risks with User Experience Engineering (Part 2)

by Dmitri Khanine

In Part 1, we introduced the idea of User Experience Engineering (UX) and the core tools and processes we use today to conduct user research. Here we continue by showing the core principle that all user research is based on--and how you can use it to combat scope creep and other requirements-based risks right now.

Divide and Direct

by Ronald B. Smith

Dividing your project into smaller parts that are more controllable helps you move closer to your ultimate goal: successfully achieving your project deliverables and high user satisfaction. Follow these seven tips to gain more direct control over your project.

Slashing Risks with User Experience Engineering

by Dmitri Khanine

What is UX, and why should you pay attention? This article will help you steer clear of common pitfalls. You'll understand the key UX activities, their goals, deliverables and what kind of outcomes you should be expecting to receive. We will also look at the degree at which each of these activities are affecting your risk breakdown structure and your schedule, their typical durations and typical manpower requirements.

The Risk-based Testing Strategy

by Patti Gilchrist, PMP

What can you do when schedules slip and there is pressure to still deliver on time? Risk-based testing starts early in the project. You should begin identifying risks to quality early--and use that awareness to guide your testing strategy.

5 Symptoms of a Sick Project

by Rob Saxon

There are few diagnostic readings that a project manager can take to determine if their project is out of control. Think of them as temperature and blood pressure. By identifying a critical mass of these readings, project managers can sooner identify problems--and change project behaviors for the better.

Testing Issues and Opportunities

by Kevin Coleman

Testing has been seen as an unavoidable evil. But as we change and adapt, we must test all of those changes to help ensure they work properly, work as designed, meet the organization’s needs and are secure.

Handling Illness: A Project Manager's Way

by Debasis Chakrabarti

A manager was suffering through a “project” in crisis--but it was not a project that he was managing at his office. The project is crisis was himself. Despite having three decades of project experience, he didn't have a risk response plan at the ready. That's when his project manager brain got to work...

Dynamic Project Management

by Pradeep Sangal

Large infrastructure project teams continuously struggle to manage several issues posed by the volatile environment of projects. It's time to develop a holistic and dynamic project planning and monitoring tool for the effective management of ever-changing project requirements and environments.

Applying Risk-Driven Project Management to Achieve Success in Complex, Innovative Projects

by Dr. Jeno Szep, PMP, CISA, CRISC, CISM, ITIL-F, Dr. Erzsebet Lugosi, PMP, CISA, CRISC, ITIL-F

In today’s rapidly changing corporate environment, innovative projects often must be completed in a very short time frame. After a thorough analysis of different methods and tools, the authors developed the Risk-Driven project management method (RiD) to facilitate the creation of complex enterprise value. Applying the 12-point maturity-value-success metric (12-point MVS metric) can help ensure proactive risk monitoring.

An Iterative Risk Management-Based Strategy for Benefits Realization

by Irfan Shariff

Project managers can facilitate benefits realization and--more strategically--the project management function. That encompasses the three Ps of project management, which can play the crucial but often missing organizational role that will embed benefits realization in strategy execution and--by extension--program and project delivery.

Managing ‘Other Duties as Assigned’

by Andy Jordan

We have all faced challenges--project work needs to be done, but it doesn’t really fit into anybody’s area of accountability. What are the unique challenges of that work, and how do project managers manage it effectively?

Cloak and Dagger Time for PMs

by Kevin Coleman

The sensitive information that is a routine part of project management is now a high-value target of corporate spies. Who said project management was boring?

Escalating Issues

by Kenneth Darter, PMP

There will come a day when you will face an issue and not be able to resolve it on your own. What will you do to ensure the project is not compromised?

A Project Lifecycle for New Hotels

by Richard Whitfield
Gert Noordzy

Addressing the problems inherent in opening new hotels, this paper advocates that hotel owners adopt a complete and well-defined project lifecycle overseen by an experienced project manager. The goals and deliverables for the key phases within this extended new hotel project lifecycle are identified and the main benefits of following this approach are explained. The proposed approach helps optimize the return on investment for each new hotel and maximizes positive cash flows from operations.

Introducing Statistical PERT

by William Davis

Isn’t there a no-cost way to create accurate, probabilistic estimates using just Microsoft Excel--without buying any add-in programs, without installing new software and without having to return to college to take a statistics class? Yes, there is: It’s called Statistical PERT.

Effective IT Leadership: What is the CIO's Role?

by Sreekumar Menon

CIOs holds the key in establishing IT as a part of the strategic business plan, which can help drive initiated changes across the organization. However, CIOs must also focus on maximizing outcomes with results in order to dramatically change the perception and actual value of IT in the eyes of the business.

Impossible Filmmaking (Part 2)

by Kevin Richardson

Nothing in his impressive experience could have prepared a time-crunched filmmaker for his hectic project in China...except one thing: earning his PMP certification. Read how this international project management consultant got an animated film off the ground in no time flat.

New Problems, Old Project

by Kenneth Darter, PMP

It never fails--at the end of the project, a whole lot of problems start cropping up. Tracking these problems in the flurry of activities occurring at the end of a project can be difficult. How can you handle these appropriately?

"I once took a cab to a drive-in. The movie cost me $190."

- Stephen Wright


“Well, it is important that people know about the work.”

“To me, the best thing about living like a lab rat, is that sometimes, really rare times, you actually discover something. On the night of a discovery, when you’re lying in bed, you are the only person in the world that knows it’s true.”

“I do believe we’ve known each other since forever.”

“Yes. You know how? When the Big Bang happened, all the atoms in the universe were all smashed together into one little dot that exploded outward, so my atoms and your atoms were certainly together then and… who knows, probably smashed together several times in the last 13.7 billion years, so my atoms have known your atoms and they’ve always known your atoms. My atoms have always loved your atoms.”

“Within our lifetimes, we’ve marveled as biologists have managed to look at ever smaller and smaller things. And astronomers have looked further and further into the dark night sky, back in time and out in space. But maybe the most mysterious of all is neither the small nor the large: it’s us, up close. Could we even recognize ourselves, and if we did, would we know ourselves? What would we say to ourselves? What would we learn from ourselves? What would we really like to see if we could stand outside ourselves and look at us?” - Another Earth (2011)

“There’s no mythology for women. If you’re reading On Poetics or whatever, the archetypal, primal stories are all about men. With women you’re either Persephone, the innocent that’s captured in the underground, or you’re Aphrodite, the unapproachable goddess. There are no gradations,” explains Brit Marling, always the charming intellectual, speaking from the Los Angeles home where she’s working on her next script. “It’s fascinating to watch women writers and directors wrestling in real time with what it means to invent the feminine narrative from scratch. Does it not look at all like the traditional, linear hero’s journey? That very classic, traditional line that we think of as storytelling. Or does the female version look different? Is it elliptical?”

 … “There are all these things around us all the time that we can sense but can’t explain,” Marling says. “Film becomes one of the beautiful tools for attempting to get at the unseen, to talk about it and to revel in it.”

Brit Marling: Inventing The Female Narrative


Ascending key columns, such as IDENTITY columns or datetime columns representing real-world timestamps, can cause inaccurate statistics in tables with frequent INSERTS because new values all lie outside the histogram.  Consider updating statistics on such columns frequently with a batch job if your application seems to be getting inadequate query plans for queries that have a condition on the ascending key column. How often to run the batch job depends on your application. Consider daily or weekly intervals, or more often if needed for your application. Alternatively, trigger the job based on an application event, such as after a bulk load or after a certain number of INSERT operations.


A differential equation is any expression that relates the values of a collection of functions and their derivatives, as well as some coefficients. One may be asked to solve (symbolically or numerically) or estimate the solution to a differential equation. Usually, they appear as the result of a mathematical model for a physical phenomenon.

Use the tag for partial differential equation questions.

history | excerpt history