Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin

Hc, Shin; Merutka, Gene; Jp, Waltho; Ll, Tennant; Dyson, H. Jane; Pe, Wright

doi:10.1021/bi00076a008

Cited by 95 publications

(85 citation statements)

References 41 publications

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“…The standard binding function, 2 H 2 O, pH 5.5, at 285 K. Trifluoroethanol (TFE) was used as a co-solvent with water because TFE-water mixtures are known to promote hydrogen bond formation within peptides that already possess an intrinsic folding capability (25). Where appropriate (for the Ad12 wild type and Ad5 E*8 peptides), deuterated dithiothreitol was added to a final concentration of 10 mM to prevent intermolecular disulfide bridge formation.…”

Section: Methodsmentioning

confidence: 99%

Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins

Molloy

Milner

Yakub

et al. 1998

Journal of Biological Chemistry

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The C-terminal binding protein (CtBP) has previously been shown to bind to a highly conserved six-amino acid motif very close to the C terminus of adenovirus early region 1A (Ad E1A) proteins. We have developed an enzyme-linked immunosorbent assay that has facilitated the screening of synthetic peptides identical or similar to the binding site on Ad E1A for their ability to bind CtBP and thus inhibit its interaction with Ad12 E1A. It has been shown that amino acids both C-terminal and N-terminal to the original proposed binding site contribute to the interaction of peptides with CtBP. Single amino acid substitutions across the binding site appreciably alter the K d of the peptide for CtBP, indicative of a marked reduction in the affinity of the peptide for CtBP. The solution structures of synthetic peptides equivalent to the C termini of both Ad5 and Ad12 E1A and two substituted forms of these have been determined by proton NMR spectroscopy. Both the Ad12 and Ad5 peptides dissolved in trifluoroethanol/water mixtures were found to adopt regular secondary structural conformations seen as a series of ␤-turns. An Ad12 peptide bearing a substitution that resulted in only very weak binding to CtBP (Ad12 L258G) was found to be random coil in solution. However, a second mutant (Ad12 V256K), which bound to CtBP rather more strongly (although not as well as the wild type), adopted a conformation similar to that of the wild type. We conclude that secondary structure (␤-turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.Adenovirus E1A DNA encodes two major proteins that are expressed from differentially spliced mRNAs of sedimentation coefficients 13 and 12 S. These two proteins are co-terminal and only differ by the presence of an extra peptide located toward the C terminus of the larger molecule. Adenovirus E1A proteins (Ad E1A) 1 are multifunctional, being able to regulate transcription of viral and cellular genes, initiate cell cycle progression and DNA synthesis (1, 2), inhibit differentiation (3, 4), and, in some cases, transform cells in culture (reviewed in Refs. 5 and 6).It is now clear that Ad E1A proteins have no enzymic properties of their own but exert their influence on the host cell through a series of protein-protein interactions with important cellular regulatory molecules. Thus, E1A has been shown to bind to pRb105 and the related p107 and p130 proteins, p300/ CBP, ATF2, and TBP (reviewed in Ref. 6). The interaction sites for all of these proteins on E1A have been established by mutational and deletional analysis, and most have been shown to occur in regions conserved in the E1As from different virus serotypes. For example, the pRb family of proteins binds in conserved regions (CRs) 1 and 2 (7, 8), whereas TBP (9 -12) and ATF2 (13-16) interact with the N-and C-terminal halves of CR3, respectively. A further region conserved between different E1As, although not generally included in the group of E1A conserved regions, occurs at the C terminus of the protein (17,1...

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Section: Methodsmentioning

confidence: 99%

Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins

Molloy

Milner

Yakub

et al. 1998

Journal of Biological Chemistry

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“…Part of the apomyoglobin molecule, consisting of the A, G, and H helices and part of the B helix, folds rapidly to form an on-pathway intermediate, with the remainder of the polypeptide chain folding at a slower rate, of the order of seconds (21,25). Peptide fragments of apomyoglobin corresponding to the G and H helices showed a propensity for helical structure (26)(27)(28), whereas peptides corresponding to the remainder of the protein showed far lower propensity for helix formation (29). Yet an experiment in which the H helix of myoglobin was mutated to decrease its helical propensity (30) showed very little influence on the folding rate of the protein.…”

Section: Experimental Verification Of Modelsmentioning

confidence: 99%

The role of hydrophobic interactions in initiation and propagation of protein folding

Dyson

Wright

Scheraga

2006

Proc. Natl. Acad. Sci. U.S.A.

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295

217

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Globular proteins fold by minimizing the nonpolar surface that is exposed to water, while simultaneously providing hydrogenbonding interactions for buried backbone groups, usually in the form of secondary structures such as ␣-helices, ␤-sheets, and tight turns. A primary thermodynamic driving force for the formation of globular structure is thus the sequestration of nonpolar groups, but the correlation between the parts of proteins that are observed to fold first (termed folding initiation sites) and the ''hydrophobicity'' (as customarily defined) of the amino acids in these regions has been quite weak. It has previously been noted that many amino acid side chains contain considerable nonpolar sections, even if they also contain polar or charged groups. For example, a lysine side chain contains four methylenes, which may undergo hydrophobic interactions if the charged -NH 3 ؉ group is saltbridged or hydrogen-bonded. Folding initiation sites might therefore contain not only accepted ''hydrophobic'' amino acids, but also larger charged side chains. Recent experiments on the folding of mutant apomyoglobins provides corroboration for models based on the hypothesis that folding initiation sites arise from hydrophobic interactions. A near-perfect correlation was observed between the areas of the molecule that are present in the burstphase kinetic intermediate and both the free energy of formation of hydrophobic initiation sites and the parameter ''average area buried upon folding,'' which pinpoints large side chains, even those containing charged or polar portions. These results provide a putative mechanism for the control of protein-folding initiation and growth by polar͞nonpolar sequence propensity alone.folding pathways ͉ myoglobin ͉ ribonuclease F reed of the ribosome, and in the absence of chaperones, a newly synthesized protein exists in an ensemble of states, the so-called statistical coil, the nature of which is a subject of much recent investigation (1). It subsequently folds to the native biologically active structure whose free energy is considered to be a minimum under appropriate solution conditions (2). Since the seminal work of Anfinsen (2), the kinetics of the folding process and the final structure have been considered to be determined by the physical interactions among the amino acid residues to attain the thermodynamically favored state.A major question has involved the exact mechanism by which the interresidue interactions specify the initiation of folding in the unfolded polypeptide chain under folding conditions. Because nonpolar groups are not soluble in water, attention has been focused on the hydrophobic interactions between nonpolar side chains to achieve their sequestration from aqueous solvent. A model has been proposed in which nearby nonpolar groups in the chain participate in hydrophobic interactions with minimal loss of entropy of the chain because of the proximity of the interacting side chains in the amino acid sequence (3).Yet the chemical nature of the polypeptide chain complicates ...

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“…The conformational ensemble of the 27-residue H-helix peptide was found to contain a high population of ordered helical forms, particularly in the center of the peptide, with fraying at the termini (Waltho et al, 1993). The 19-residue G-helix peptide showed only marginal preferences for helix formation in water, but was folded to a highly helical state at low concentration (20% v/v) of the helix-promoting solvent TFE (Shin et al, 1993a). In addition, the 5-residue GH-turn peptide was found to populate a turn conformation in aqueous solution (Shin et al, 1993b).…”

Section: Implications For Initiation Of Apomyoglobin Foldingmentioning

confidence: 99%

Folding propensities of peptide fragments of myoglobin

et al. 1997

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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,

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Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin

Cited by 95 publications

References 41 publications

Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins

Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins

The role of hydrophobic interactions in initiation and propagation of protein folding

Folding propensities of peptide fragments of myoglobin

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