The cytoplasmic fragment of the aspartate receptor displays globally dynamic behavior

A number of cloned soluble fragments if the bacterial chemotaxis transmembrane receptors retain partial function. Prior studies of a fragment corresponding to the cytoplasmic domain (c-fragment) of the Escherichia coli aspartate receptor have correlated the signaling state of mutant receptors with the oligomerization state of the c-fragments: equilibria of smooth-swimming mutants are shifted toward oligomeric states; tumble mutants are shifted toward monomeric states [Long, D. G., & Weis, R. M. (1992) Biochemistry 31, 9904-9911]. We have applied several experimental probes of local and global structural flexibility to two signaling states, the wild-type (monomeric) and S461L smooth mutant (predominantly dimeric) c-fragments. Featureless near-UV CD spectra are observed, which indicate that the single Trp residue is in a symmetric environment (most likely averaged by fluctuations) and suggest that the C-termini of both proteins are highly mobile. Both proteins undergo extremely rapid proteolysis and enhance ANS fluorescence, which indicates that many sites are accessible to trypsin cleavage and hydrophobic sites are accessible to ANS binding. The global nature of the flexibility is demonstrated by 1H NMR studies. Lack of chemical shift dispersion suggests that fluctuations average the environments of side chains and backbone protons. Rapid exchange of 99% of the observable amide protons suggests that these fluctuations give high solvent accessibility to nearly the entire backbone. This evidence indicates that both monomeric and dimeric c-fragments are globally flexible proteins, with properties similar to "molten-globule" states. The significance of this flexibility depends on whether it is retained in functioning receptors: the c-fragment structure may lack important tertiary contacts, protein-protein interactions, or topological constraints needed to stabilize a nondynamic native structure, or the cytoplasmic domain of the native receptor may retain flexibility which may be modulated in the mechanism of transmembrane signaling.