Conformational features of a hexapeptide model Ac-TGAAKA-NH2 corresponding to a hydrated alpha helical segment from glyceraldehyde 3-phosphate dehydrogenase: implications for the role of turns in helix folding.

Recent analysis of alpha helices in protein crystal structures, available in literature, revealed hydrated alpha helical segments in which, water molecule breaks open helix 5-->1 hydrogen bond by inserting itself, hydrogen bonds to both C=O and NH groups of helix hydrogen bond without disrupting the helix hydrogen bond, and hydrogen bonds to either C=O or NH of helix hydrogen bond. These hydrated segments display a variety of turn conformations and are thought to be 'folding intermediates' trapped during folding-unfolding of alpha helices. A role for reverse turns is implicated in the folding of alpha helices. We considered a hexapeptide model Ac-1TGAAKA6-NH2 from glyceraldehyde 3-phosphate dehydrogenase, corresponding to a hydrated helical segment to assess its role in helix folding. The sequence is a site for two 'folding intermediates'. The conformational features of the model peptide have been investigated by 1H 2D NMR techniques and quantum mechanical perturbative configuration interaction over localized orbitals (PCILO) method. Theoretical modeling largely correlates with experimental observations. Based upon the amide proton temperature coefficients, the observed d alpha n(i, i + 1), d alpha n(i, i + 2), dnn(i, i + 1), d beta n(i, i + 1) NOEs and the results from theoretical modeling, we conclude that the residues of the peptide sample alpha helical and neck regions of the Ramachandran phi, psi map with reduced conformational entropy and there is a potential for turn conformations at N and C terminal ends of the peptide. The role of reduced conformational entropy and turn potential in helix formation have been discussed. We conclude that the peptide sequence can serve as a 'folding intermediate' in the helix folding of glyceraldehyde 3-phosphate dehydrogenase.

Conformational features of reduced and disulfide intact forms of hen egg white lysozyme in aqueous solution in presence of 3-chloro-1, 2-propanediol and dioxane: implications for protein folding intermediates.

Conformational features of reduced and disulfide intact hen egg white lysozyme in aqueous 1,4-dioxane and 3-chloro-1, 2-propanediol solutions have been examined using circular dichroism and fluorescence spectroscopy. We find that in presence of 1, 4-dioxane, reduced lysozyme assumes a relatively compact conformational form with secondary structure closer to native state and no tertiary structure as judged by peptide and aromatic CD spectra and ANS binding studies monitored by fluorescence. Further, in presence of 40% (v/v) 3-chloro-1, 2-propanediol, disulfide intact lysozyme (DI-lysozyme) assumes a conformational form with native like secondary structure and no tertiary structure akin to a molten globule state. We correlate our results to kinetic hydrogen- deuterium exchange NMR results of the refolding of lysozyme available in literature and suggest that the conformational forms observed in our study could be models for kinetic intermediates in the refolding of lysozyme.

Conformational features of a peptide model Ac-DTVKLMYKGQPMTFR-NH2, corresponding to an early folding beta hairpin region of staphylococcal nuclease.

Recent H-D exchange 1H NMR studies of the refolding of Staphylococcal nuclease (P117G) variant suggest that, a region of the protein corresponding to a beta hairpin in the native structure folded early in the refolding process. In order to investigate whether the formation of beta hairpin is an early folding event, we investigated the conformational features of the beta hairpin peptide model Ac-DTVKLMYKGQPMTFR-NH2 from Staphylococcal nuclease with 1H NMR techniques. It appears that the peptide aggregates even at a low concentration. However, based on the observation of weak dnn(i, i + 1) NOEs between K8-G9, G9-Q10, an upfield shift of Gly9 NH and a low temperature coefficient (-d delta/dT) for Gly9 NH, we suggest that the sequence YKGQP as part of the beta hairpin peptide model samples conformational forms with reduced conformational entropy and turn potential. The presence of aggregation could be restricting the population of folded conformational forms and formation of beta hairpin at detectable concentrations. We suggest that, formation of beta hairpin could be an early event in the folding of Staphylococcal nuclease and this observation correlates with H-D exchange 1H NMR results and also with the prediction of a protein folding model proposed in literature.

Conformational preference of Leu side chain in melanostatin in DMSO.

The solution conformation of melanostatin (Pro-Leu-Gly-NH2) in the neutral and protonated forms of DMSO has been monitored by one and two dimensional NMR techniques at 500 MHz. The temperature coefficients of the amide proton chemical shifts in conjunction with the observed NOESY spectra suggest that melanostatin in neutral form in DMSO adopts a backbone conformation such that leucine amide proton is buried by the proline ring and the side chain of leucine. Similar observation is made for protonated form of melanostatin in DMSO. The results of the present study are at variance with the earlier NMR studies which proposed a beta-turn structure for both the forms of melanostatin. There is, however, no evidence for the presence of beta-turn structure for both the forms of melanostatin in DMSO. In CDCl3 also Leu NH appears to be buried as evident from the solvent titration with DMSO and NOESY spectra.

A pentapeptide model for an early folding step in the refolding of staphylococcal nuclease: the role of its turn propensity.

Recently the folding of a staphylococcal nuclease (P117G) variant was examined with the hydrogen-deuterium (H-D) exchange technique. Many of the residues that showed significant protection are located in beta-sheet regions. About half the residues protected belong to an antiparallel beta-hairpin structure (residues 21-35) in the native structure. The beta-hairpin structure is formed by strands 2 and 3 of sheet 2 connected by the sequence 27Y KGQP31 in a type 1' reverse turn conformation with a 4-->1 hydrogen bonding between Q30 NH and Y27 C = O. We have targeted the conformational characterization of the peptide model Ac-YKGQP-NH2 with 1H two-dimensional nmr techniques in aqueous solution with a view to assessing its propensity to sample turn conformational forms and thus initiate the formation of beta-hairpin structure. Based upon the observed d alpha n (i, i + 1), d alpha n (i, i + 3), and dnn (i, i + 1) nuclear Overhauser effect connectivities, temperature coefficients for amide protons and conformational analysis with quantum mechanical perturbative configuration interaction over localized orbitals method, we conclude that the model peptide samples turn conformational forms with reduced conformational entropy. We suggest that the turn can nucleate the formation of the beta-hairpin structure in the refolding of nuclease. Observation of turn propensity for this sequence is consistent with the folding mechanism of the Greek key motif (present in Staphylococcal nuclease) proposed in the literature.

Harnessing D-amino acids for peptide motif designs. Synthesis and solution conformation of Boc-D-Glu-Ala-Gly-Lys-NHMe and Boc-L-Glu-Ala-Gly-Lys-NHMe.

In examining the use of D-amino acids in designing specific peptide folding motifs, the tetrapeptide Boc-D-Glu-Ala-Gly-Lys-NHMe 1 and its analog 2 featuring L-Glu were synthesized for a comparison of their solution conformations by NMR spectroscopy. The temperature coefficients of amide proton resonances, NOE data, side-chain CH2 anisotropies and salt titration results suggest a weak type II reverse-turn conformation for peptide 2, and a tandem II' turn-3(10)-helix conformation of appreciable conformational stability for peptide 1 in apolar solvents. The latter is of potential interest as the N-terminal helix cap that could support the design of longer 3(10) helices. Possible origins of appreciable difference in the conformational stabilities of the diastereomers are discussed.