C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1C, pH 5, 0.1 M NaCI), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9 His-12+ stabilizes the helix. Formationofthehelixisenthalpydriven [van'tHoff AH, -16kcal/ mol (1 cal = 4.18 J)] and the helix is not observed above 30C.Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short at-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize a-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of a-helices in proteins.Short a-helices, of the size range usually found in globular proteins (6-20 residues), are highly unstable in water in the absence of specific stabilizing interactions, according to data obtained with random copolymers by the "host-guest" technique (1). For short helices (n < 20) and for o << 1, the ratio ofhelical to nonhelical residues depends on the quantity 0,rn-l/(S -1)2 (see Note Added in Proof), where o is the helix nucleation constant, s is an average stability constant for one residue, and n is the number of H-bonded residues in the helix. Values of s vary from 0.5 to 1.3 for different amino acid residues at 0-60TC, while o is estimated to be~-'10`(1), so that the fraction of molecules in helical form is expected to be 10`for a short helix. Most studies of helix formation by protein fragments agree with this deduction. The three cyanogen bromide peptides of sperm whale myoglobin give CD spectra that indicate that very little a-helix is present in water at 250C, although partly helical spectra are obtained in 95% methanol (2). The conclusion has been drawn that tertiary interactions are needed to stabilize the a-helices of globular proteins in water.An exception was found by Brown and Klee (3): C-peptide of RNase A has a CD spectrum indicative of partial helix formation in water at 10C. They found that the helix forms monomolecularly, is unstable at 260C, and is unstable in deionized water. Molecular weight measurements by sedimentation equilibrium showed that C-peptide is monomeric at concentrations up to 1 mM under conditions in which helix formation occurs (10C; ionic strength, 0.1 M). Freedom from aggregation at moderate concentration is an important prop...

A BSTR ACTA 12-residue peptide AcDKDGDGY-ISAAENH 2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K ‫؍‬ 10 5 M ؊1 ). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the ␣-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH 2 presented in this paper show that residues A13-Q16 form an ␣-helix of very high stability when the La 3؉ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1°C and 70% at 25°C. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent ␣-helices is discussed. The problem of CD spectra of very short ␣-helices is also addressed.

S100A1 is a typical representative of a group of EF‐hand calcium‐binding proteins known as the S100 family. The protein is composed of two α subunits, each containing two calcium‐binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca2+‐titration curves of Trp90 fluorescence for both the native protein and its Glu32 → Gln mutant with an inactive N‐loop. Using a chelator method, we also determined the calcium‐binding constant for the S100A1 Glu73 → Gln mutant with an inactive C‐loop. The protein binds four calcium ions in a noncooperative way with binding constants of K1 =4 ± 2 × 103 m−1 (C‐loops) and K2≈ 102 m−1 (N‐loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2‐p‐toluidinylnaphthalene‐6‐sulfonic acid – a fluorescent probe of protein‐surface hydrophobicity. S‐Glutathionylation of the single cysteine residue (85) of the α subunits leads to a 10‐fold increase in the affinity of the protein C‐loops for calcium and an enormous – four orders of magnitude – increase in the calcium‐binding constants of its N‐loops, owing to a cooperativity effect corresponding to ΔΔG = −6 ± 1 kcal·mol−1. A similar effect is observed upon formation of the mixed disulfide with cysteine and 2‐mercaptoethanol. The glutathionylated protein binds TRTK‐12 peptide in a calcium‐dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.

S100 proteins play a crucial role in multiple important biological processes in vertebrate organisms acting predominantly as calcium signal transmitters. S100A1 is a typical representative of this family of proteins. After four Ca(2+) ions bind, it undergoes a dramatic conformational change, resulting in exposure, in each of its two identical subunits, a large hydrophobic cleft that binds to target proteins. It has been shown that abnormal expression of S100A1 is strongly correlated with a number of severe human diseases: cardiomyopathy and neurodegenerative disorders. A few years ago, we found that thionylation of Cys 85, the unique cysteine in two identical S100A1 subunits, leads to a drastic increase of the affinity of the protein for calcium. We postulated that the protein activated by thionylation becomes a more efficient calcium signal transmitter. Therefore, we decided to undertake, using nuclear magnetic resonance methods, a comparative study of the structure and dynamics of native and thionylated human S100A1 in its apo and holo states. In this paper, we present the results obtained for both forms of this protein in its holo state and compare them with the previously published structure of native apo-S100. The main conclusion that we draw from these results is that the increased calcium binding affinity of S100A1 upon thionylation arises, most probably, from rearrangement of the hydrophobic core in its apo form.