  Thermophilic enzymes can operate at higher temperatures but show reduced
activities at room temperature. They are in general more stable during
preparation and, accordingly, are considered to be more rigid in structure.
Crystallization is often easier compared to proteins from bacteria growing at
ambient temperatures, especially for membrane proteins. The ATP-producing
enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum
strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We
applied a single-molecule F\"orster resonance energy transfer (FRET) approach
using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor
the expected 13-stepped rotary Fo motor at work. New FRET transition histograms
were developed to identify the smaller step sizes compared to the 10-stepped Fo
motor of the Escherichia coli enzyme. Dwell time analysis revealed the
temperature and the LDAO dependence of the Fo motor activity on the single
molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating
a high flexibility in the membrane part of this thermophilic enzyme.
