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The F helix region of sperm whale apomyoglobin (apoMb) is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. In order to examine the effects of F helix stabilization on the folding pathway of apoMb, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared, a double mutant P88K/S92K (F2) and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B-helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the G-H turn region in the G23A/G25A mutant, suggesting that stabilization of the B-helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.

Elongation factor Tu (EF-Tu) promotes binding of aminoacyl-tRNA to the A site of the ribosome. Here, we report the effects of mutations in helix D of EF-Tu and in the C-terminal domain of L7/12 on the kinetics of A-site binding. Reaction rates were measured by stopped-flow and quench-flow techniques. The rates of A-site binding were decreased by mutations at positions 144, 145, 148, and 152 in helix D of EF-Tu as well as at positions 65, 66, 69, 70, 73, and 84 in helices 4 and 5 of L7/12. The effect was due primarily to the lower association rate constant of ternary complex binding to the ribosome. These results suggest that helix D of EF-Tu is involved in an initial transient contact with helices 4 and 5 of L7/12 that promotes ternary complex binding to the ribosome. By analogy to the interaction of helix D of EF-Tu with the N-terminal domain of EF-Ts, the contact area is likely to consist of a hydrophobic patch flanked by two salt-bridges.

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.

Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L18 on Escherichia coli 5 S RNA using both end-labelling and reverse transcriptase procedures. The results, together with earlier data, were superimposed on a cylindrical projection of RNA double helices and most of the protection effects were found to cluster in the major groove at two sites located on one side of the RNA at the junctions of helix II with the adjoining internal loops A and B. The loop A/helix II junction was investigated using 5 S RNA mutants, produced by site-directed mutagenesis, that exhibited altered binding properties to L18. These results, together with those from a circular dichroism study of L18 complexed with the wild-type and different mutant RNAs, enabled us to assign an L18-induced conformational change to loop A. We infer that this change contributes to the co-operative binding of L5 to helix I, which may be reinforced by the binding of the very basic N-terminal peptide of L18 within the minor groove of helix I. A psoralen derivative formed a mono-addition product with U25 within loop B in the free RNA but not in the L18 complex. Moreover, the modified molecules were selected against in L18 binding experiments. Protection effects that occurred within the adjoining helix III and loop C were compatible with a tertiary interaction between loop C and loop B/helix III that could be stabilized by the L18 binding to the junction of helix II and loop B. Further support for a bipartite binding site derived from the finding that ethidium bromide molecules that are displaced from E. coli 5 S RNA by L18 intercalate both at the loop A/ helix II junction and in loop B at the binding site of the psoralen derivative.

In this report we describe a general survey of all helices found in 57 of the known protein crystal structures, together with a detailed analysis of 48 alpha-helices found in 16 of the structures that are determined to high resolution. The survey of all helices reveals a total of 291 alpha-helices, 71 3(10)-helices and no examples of pi-helices. The conformations of the observed helices are significantly different from the "ideal" linear structures. The mean phi, psi angles for the alpha- and 3(10)-helices found in proteins are, respectively, (-62 degrees, -41 degrees) and (-71 degrees, -18 degrees). A computer program, HBEND, is used to characterize and to quantify the different types of helix distortion. alpha-Helices are classified as regular or irregular, linear, curved or kinked. Of the 48 alpha-helices analysed, only 15% are considered to be linear; 17% are kinked, and 58% are curved. The curvature of helices is caused by differences in the peptide hydrogen bonding on opposite faces of the helix, reflecting carbonyl-solvent/side-chain interactions for the exposed residues, and packing constraints for residues involved in the hydrophobic core. Kinked helices arise either as a result of included proline residues, or because of conflicting requirements for the optimal packing of the helix side-chains. In alpha-helices where there are kinks caused by proline residues, we show that the angle of kink is relatively constant (approximately 26 degrees), and that there is minimal disruption of the helix hydrogen bonding. The proline residues responsible for the kinks are highly conserved, suggesting that these distortions may be structurally/functionally important.