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Notes: MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.

Notes: The bond energy constant of methionine Sδ—Cε, 170.066 kcal mol−1 Å−2, is given as a default value in X-ray protein structure refinement with X-PLOR [Brünger (1992). X-PLOR Version 3.1. A system for X-ray Crystallography and NMR. New York University Press]. When the atomic parameters of 3564 amino acid residues of bovine heart cytochrome c oxidase were refined at 2.0 Å resolution by using X-PLOR with default restraining parameters, 36 bond lengths deviated by over 0.06 Å from their ideal values. Out of the 36 bonds, 25 were methionine Sδ—Cε bonds. Refinement with an energy parameter of 500.0 kcal mol−1 Å−2 for the methionine Sδ—Cε bond resulted in convergence of the Sδ—Cε bond lengths to within 0.06 Å from their ideal values and reduced the crystallographic R and free-R factors by 0.6 and 0.3%, respectively. Consequently, a strong bond energy constant for Sδ—Cε of 500.0 kcal mol−1 Å−2 is recommended instead of the default value of 170.066 kcal mol−1 Å−2.