An important issue in understanding the relationship between protein sequence and structure is the degree to which different amino acids favour the formation of particular types of secondary structure. Estimates of the 'helix-forming tendency' of amino acids have been made based on 'host-guest' experiments, in which copolymers are made of the amino acid of interest (the 'guest') and a host residue (typically hydroxypropyl- or hydroxybutyl-L-glutamine). Recently, however, short alanine-based peptides were found to form stable monomeric helices in water, contrary to the result predicted from host-guest experiments. We have now measured the helix-forming tendency of five different nonpolar amino acids (Ala, Ile, Leu, Phe, Val) by substituting each in turn for alanine in a 17-residue alanine-based peptide and determining the extent of alpha-helix formation. Our results differ from those of host-guest experiments both in the degree of variation in helix-forming tendency of different amino acids, and in the rank order of the helix-forming tendency. We conclude that the helix-forming tendency of a particular amino acid depends on the sequence context in which it occurs; and the restriction of side-chain rotamer conformations is important in determining the helix-forming tendency.

Peptides of the sequence Ac-XKAAAAKAAAAKAAAAK-amide, where X is Tyr, Trp, or Ala, produce circular dichroism spectra that are typical of the a-helix; there are, however, significant differences between the Tyr, Trp, or Ala peptides in the magnitudes of the far-ultraviolet bands. A tyrosine or tryptophan residue is needed in each peptide in order to measure accurately the peptide concentration and the mean residue ellipticity. The N-or C-terminal position is chosen because helix fraying is greatest at each end and the 'Tyr or Trp residue should influence the helix content of the peptide least at these positions. Amide proton exchange measurements by proton nuclear magnetic resonance spectroscopy indicate that the Tyr, Trp, and Ala peptides possess similar amounts of H-bonded secondary structure. Comparison of the far-ultraviolet circular dichroism and absorption spectra of these peptides suggests that the differences in circular dichroism arise in each case from an induced aromatic circular dichroism band, which is positive for Tyr and negative for Trp. Insertion of one to three Gly residues between the aromatic residue and the rest of the helical sequence reduces the induced aromatic band to insignificant levels. Using this procedure of inserting Gly residues between the Tyr and the rest of the helical sequence, we remeasured the helix propensity of Gly. We find that the A1a:Gly ratio of helix propensities is 40, as opposed to our previous estimate of 100 determined using the Tyr peptide without considering the aromatic contribution of Tyr in the analysis [Chakrabartty, A., Schellman, J.

SummaryPhotoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here, we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide a visualization of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter −35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation.

Cobalamin (B 12 ) typically functions as an enzyme cofactor but can also regulate gene expression via RNA-based riboswitches. B 12 -directed gene regulatory mechanisms via protein factors have, however, remained elusive. Recently, we reported down-regulation of a light-inducible promoter in the bacterium Myxococcus xanthus by two paralogous transcriptional repressors, of which one, CarH, but not the other, CarA, absolutely requires B 12 for activity even though both have a canonical B 12 -binding motif. Unanswered were what underlies this striking difference, what is the specific cobalamin used, and how it acts. Here, we show that coenzyme B 12 (5′-deoxyadenosylcobalamin, AdoB 12 ), specifically dictates CarH function in the dark and on exposure to light. In the dark, AdoB 12 -binding to the autonomous domain containing the B 12 -binding motif foments repressor oligomerization, enhances operator binding, and blocks transcription. Light, at various wavelengths at which AdoB 12 absorbs, dismantles active repressor oligomers by photolysing the bound AdoB 12 and weakens repressor-operator binding to allow transcription. By contrast, AdoB 12 alters neither CarA oligomerization nor operator binding, thus accounting for its B 12 -independent activity. Our findings unveil a functional facet of AdoB 12 whereby it serves as the chromophore of a unique photoreceptor protein class acting in light-dependent gene regulation. The prevalence of similar proteins of unknown function in microbial genomes suggests that this distinct B 12 -based molecular mechanism for photoregulation may be widespread in bacteria.carotenogenesis | Thermus thermophilus | antirepressor | MerR | TtCarH