Folding propensities of peptide fragments of myoglobin

Abstract

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthesized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformational preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the a region of (c#J,$) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A-and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate p-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, earlier studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.Keywords: myoglobin; nuclear magnetic resonance; peptide conformation; protein folding; reverse turn; secondary structureThe mechanism by which proteins fold into their native conformations remains one of the central unsolved problems in molecular biology. One view is that folding occurs along defined pathways, often with structured intermediates (for reviews, see Creighton,

Dynamic folding pathway models of the villin headpiece subdomain (HP‐36) structure

“…of native contacts (tertiary structure), with the correlation coefficient of 0.94. Thus, as far as the correlation between the formation of the secondary structures and the completion of the tertiary structure is concerned the present folding model is consistent with both computational 3,4 and experimental 43 results. The current study also agrees with the computational work by Duan et al, 3 where it is shown that the burst phase precedes the slow conformational readjustment phase in HP-36 folding.…”

“…This observation that the secondary structure is formed in the presence of partial tertiary structure, is consistent with experimental observation. 43 ADMD pathway for HP-36 is consistent with the hydrophobic collapse model 44 in that the tertiary and secondary structures are formed concurrently after the initial hydrophobic collapse. After the collapse (i.e., j > 950), we find a good correlation between the number of native hydrogen bonds (secondary structure) and the number Figure 6.…”

“…This is probably due to the low intrinsic helical stability/propensity of the three helices in HP-36 when exposed in water. 43,46 One may want to identify the transition state along the ADMD folding pathway obtained in this work, for which free energy profile projected on a well-defined reaction coordinate should be calculated. Although the ADMD formalism does not provide direct ways to obtain free energy profiles, one may consider to perform Monte Carlo/molecular dynamics simulations to estimate the free energy difference between successive conformations along the pathway, which is beyond the scope of this work.…”

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

“…Studies of isolated peptide fragments of apomyoglobin indicate that the H helix possesses the highest propensity for spontaneous formation of helical structure in aqueous solution, whereas the fragments corresponding to the G helix possess poor helix propensity in isolation (24,25). Both the G and A helices show extensive helix formation in longer peptides where interhelix contacts are possible (26). These data clearly show the importance of tertiary interactions for folding of the A and G helices.…”

The early folding kinetics of apomyoglobin

“…To further this understanding, the kinetic aspects of the folding of apomyoglobin have been the subject of considerable experimental study (Hughson et al, 1990(Hughson et al, , 1991Jennings & Wright, 1993; Shin et al, 1993a Shin et al, , 1993bWaltho et al, 1993;Jennings et al, 1994;Eliezer et al, 1995; Loh et al, 1995;Ballew et al, 1996;Reymond et al, 1997). Elucidating the kinetic aspects of folding requires information on how the peptide elements that make up the protein interact dynamically to produce the native folding pattern.…”