Maureen Pitkeathly

The effects of guanidine hydrochloride on the 'random coil' conformations and NMR chemical shifts of the peptide series GGXGG.

The effects of the commonly used denaturant guanidine hydrochloride(GuHCl) on the random coil conformations and NMR chemical shifts of theproteogenic amino acids have been characterized using the peptide seriesAc-Gly-Gly-X-Gly-Gly-NH2. The φ angle-sensitive couplingconstants, ROESY cross peak intensities and proline cis–trans isomerratios of a representative subset of these peptides are unaffected by GuHCl,which suggests that the denaturant does not significantly perturb intrinsicbackbone conformational preferences. A set of3JHNHα values is presented which agreewell with predictions of recently developed models of the random coil. Wehave also measured the chemical shifts of all 20 proteogenic amino acids inthese peptides over a range of GuHCl concentrations. The shifts exhibit alinear dependence on denaturant concentration and we report here correctionfactors for the calculation of ‘random coil’ 1H chemicalshifts at any arbitrary denaturant concentration. Studies of arepresentative subset of peptides indicate that 13C and15N chemical shifts are also perturbed by the denaturant.These results should facilitate the application of chemical shift-basedanalytical techniques to the study of polypeptides in solution with GuHCl.The effects of the denaturant on the quality of NMR spectra and on chemicalshift referencing are also addressed.

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.

Solution structure of a peptide fragment of human α–lactalbumin in trifluorethanol: a model for local structure in the molten globule

BACKGROUND At low pH, human alpha-lactalbumin forms a partly folded molten globule state that contains a non-native clustering of the side chains of Tyr103, Trp104 and His107. In order to understand the conformation of this region of the protein in the molten globule state, we investigated the structure of a peptide corresponding to residues 101-110 of human alpha-lactalbumin in trifluoroethanol. RESULTS We determined the structure of the 101-110 peptide from an NMR data set of 145 nuclear Overhauser effects and nine 3JHN alpha coupling constants, using an ensemble calculation approach to take into account the possibilities of conformational averaging of the data. The backbone of residues 3-10 in the peptide adopts a series of turns, that involving residues 5-8 being the best defined, while the side chains of residues 1, 3, 4, 5, 6 and 7 form a hydrophobic cluster. CONCLUSIONS The peptide conformation differs from that previously determined for residues 101-110 in crystal structures of native alpha-lactalbumin determined at both high and low pH, particularly in the relative orientations of the side chains. The series of turns seen in the peptide could, however, be related to the alpha-helical structure seen for residues 104-111 in crystals at high pH, and may be important in the molten globule state for bringing the peptide chain into a compact conformation where favourable interactions between the side chains can occur.

Native-like secondary structure in a peptide from the α-domain of hen lysozyme

Background: To gain insight into the local and nonlocal interactions that contribute to the stability of hen lysozyme, we have synthesized two peptides that together comprise the entire α -domain of the protein. One peptide (peptide 1–40) corresponds to the sequence that forms two α -helices, a loop region, and a small β -sheet in the N-terminal region of the native protein. The other (peptide 84–129) makes up the C-terminal part of the α -domain and encompasses two α -helices and a 3 10 helix in the native protein. Results: As judged by CD and a range of NMR parameters, peptide 1–40 has little secondary structure in aqueous solution and only a small number of local hydrophobic interactions, largely in the loop region. Peptide 84–129, by contrast, contains significant helical structure and is partially hydrophobically collapsed. More specifically, the region corresponding to helix C in native lysozyme is disordered, whereas regions corresponding to the D and 3 10 helices in the native protein are helical in this peptide. The structure in peptide 84–129 is at least partly stabilized by interactions between residues in the two helical regions, as suggested by further NMR analysis of three short peptides corresponding to the individual helices in this region of the native protein. Conclusions: Stabilization of structure in the sequence 1–40 appears to be facilitated predominantly by long-range interactions between this region and the sequence 84–129. In native lysozyme, the existence of two disulphide bonds between the N- and C-terminal halves of the α -domain is likely to be a major factor in their stabilization. The data show, however, that native-like secondary structure can be generated in the C-terminal portion of the α -domain by nonspecific and nonnative interactions within a partially collapsed state.

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association.

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of1H-15N-correlated 2D HSQC,15N-edited 3D TOCSY-HMQC, and15N-edited 3D NOESY-HMQC spectra. By analysis of the α-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.

α and β Conformational preferences in fibril forming peptides characterised using NMR and CD techniques

Peptide fragments taken from residues 18‒54 of short consensus repeat 3 (SCR3) from the human complement receptor CR1 have been found in aqueous solution to slowly aggregate and form fibrils. NMR studies of the monomeric form of these peptides show that they are essentially unfolded in aqueous solution and that they all have an increased helicity in TFE solutions. The behaviour of residues 28‒31 from SCR3 is particularly interesting. These residues have a high β-sheet propensity in the native protein and a seven peptide containing their sequence is found to form fibrils despite its short length. However, NMR studies show that these residues adopt a well-defined α-helix in 80% TFE and under these conditions fibril formation has not been observed. These data demonstrate the strong dependence of conformational propensities on environment.