Analysis of short interproton distances in proline peptides as a guide in the interpretation of nuclear overhauser effects

The conformational dependence of interproton distances in model proline peptides has been investigated in order to facilitate interpretation of the results of Nuclear Overhauser Effect (NOE) studies on such peptides. For this purpose two model systems, namely, Ac-Pro-NHMe and Ac-Pro-X-NHMe have been chosen and used. In the former, short interproton distances detectable in NOE experiments permit a clear distinction between conformations with Pro ψ = -300 (helical region) and those in which ψ is around 1200 (polyproline region). For the latter, the variation of distances between the protons of methyl amide and the Pro ring have been studied by superimposing on the Ramachandran map in the (φ3, ψ3) plane. The results show that β-turns and non-β-turn conformations can be readily distinguished from NOE data and such long range NOEs should be detectable for specific non-β-turn conformations. NOEs involving Cβ and Cγ protons are particularly sensitive to the state of pyrrolidine ring puckering

Analysis of short interproton distances in proline peptides as a guide in the interpretation of nuclear overhauser effects

The conformational dependence of interproton distances in model proline peptides has been investigated in order to facilitate interpretation of the results of Nuclear Overhauser Effect (NOE) studies on such peptides. For this purpose two model systems, namely, Ac-Pro-NHMe and Ac-Pro-X-NHMe have been chosen and used. In the former, short interproton distances detectable in NOE experiments permit a clear distinction between conformations with Pro ψ=−30° (helical region) and those in which ψ is around 120° (polyproline region). For the latter, the variation of distances between the protons of methyl amide and the Pro ring have been studied by superimposing on the Ramachandran map in the (φ<SUB>3</SUB>, ψ<SUB>3</SUB>) plane. The results show that β-turns and non-β-turn conformations can be readily distinguished from NOE data and such long range NOEs should be detectable for specific non-β-turn conformations. NOEs involving C<SUP>β</SUP>and C<SUP>γ</SUP> protons are particularly sensitive to the state of pyrrolidine ring puckering

Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with ω−β′\omega-\beta′ interaction

Omega $(\omega)$, consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of \omega in which it helps to maintain \beta' in a correct conformation and recruit it to the $\alpha_2 \beta$ subassembly to form a functional core enzyme $(\alpha_2 \beta \beta'\omega)$. The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of \omega latch onto the N-terminal and C-terminal ends of the \beta'-subunit. In the present study we have monitored the conformational changes in \beta' as the denatured protein is refolded in the presence and absence of \omega using tryptophan fluorescence emission of \beta' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of \omega is helpful in \beta' refolding. We have also monitored the behavior of the C-terminal tail of \omega by engineering three cysteine residues at three different sites in \omega and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of \omega is mobile in the free protein and gets restrained in the presence of \beta'. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the \beta' subunit whereas the end of the C-terminal of \omega, although it is restrained in the presence of \beta', has no interacting partner within a 4-Å radius

Analysis of short loops connecting secondary structural elements in proteins

The recognition by Ramachandran et al. (1) that the range of polypeptide chain conformation is limited by steric constraints constitutes the first step in defining the limits of the problem of protein folding. The srystal structures of globular proteins reveal intricate arrays of secondary structure modules, separated by irregular elements of the polypeptide chain termed as loops, which are uniquely assembled to yield the native folded conformation (2,3). While much early work in the area of protein structure analysis focused on the regular elements of the structure, it is becoming increasingly apparent that the less regular segments or loops merit detailed study (4-10). Protein engineering appeoaches which permit transfer of loops from one context to another or allow truncation of connecting elements (11-13), have provided a fresh impetus to the present study, short loops (&#8804;5 residues) connecting secondary structure modules are identitied from a data set of 65, largely non-homologous, protein crystal structures. Four types of super-secondary structural motifs involving &#945;-helices and b-strands viz. &#945;&#945;, &#946;&#946;, &#945;&#946; and &#946;&#945; are considered and the conformational and composotional properties of the connecting loops are examined. An analysis of the spatial orientation of the two linked secondary structural elements reveals reasonably frequent occurrence of an 'L'-shaped motif involving two orthogonally disposed B-strands