Effect of central-residue replacements on the helical stability of a monomeric peptide

Merutka, Gene; Lipton, William; Shalongo, William; Park, Soon Ho; Stellwagen, Earle

doi:10.1021/bi00484a021

Cited by 128 publications

(75 citation statements)

References 22 publications

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“…The results therefore also supported the idea that alanine is a residue of high helix propensity (Marqusee et al, 1989; Dao-pin et al, 1990; Lyu et al, 1990; Merutka et al, 1990; O'Neil & DeGrado, 1990). It was also noted in the prior study that replacement of the buried residue Leu 133 with alanine was substantially destabilizing.…”

supporting

confidence: 86%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within a-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the a-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions.All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest ( 5 0 . 4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133 +Ala made a-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino-and carboxyterminal domains changed by 3.6". This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.Keywords: alanine; lysozyme; protein folding; protein structure; thermostability In a systematic attempt to identify residues important in the folding and stability of phage T4 lysozyme we previously substituted four alanines within the a-helix that includes residues 126-134 . The helix is amphipathic, located on the surface of the carboxy-terminal domain, and is remote from the active site ( Fig. 1; Kinemage 1). In wild-type lysozyme this a-helix already includes three alanines. It was found that substitution of three solvent-exposed residues, Glu 128, Val 131, and Asn

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supporting

confidence: 86%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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“…It should be noted, however, that Miick and coworkers (1992) have evidence suggesting that alanine-based peptides form mixed 3,0-and a-helical conformations. Peptides with these same general sequences have been used to investigate the helix-stabilizing properties of different ion pairs (Merutka & Stellwagen, 1991) and also to measure the relative helix propensities of all 20 amino acids (Merutka et al, 1990). Scholtz et al (1991a) determined the parameters of helix-coil transition theory using peptides with an (AEAAKA), repeating sequence and chain lengths varying from 14 to 50 residues.…”

Section: Properties Of Helix Formationmentioning

confidence: 99%

Helical peptides with three pairs of Asp‐Arg and Glu‐Arg residues in different orientations and spacings

1993

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The helix-stabilizing effects of repeating pairs of Asp-Arg and Glu-Arg residues have been characterized using a peptide system of the same design used earlier to study Glu-Lys . The consequences of breaking ion pair and charge-helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA.The results are as follows: (1) Remarkably, both Asp-Arg and Glu-Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu-Lys and Asp-Lys peptides:The ion pairs and charge-helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability. Because the four peptides of each set have nearly identical compositions, the large differences in helix content within each set cannot be caused by differences in length and sequence. (3) Significant differences in helix content remain at 5 M NaCI, where ion pair interactions are completely screened. The residual interactions may either be nonscreenable H-bond interactions or remaining charge-helix dipole interactions. Keywords: a-helices; charge-helix dipole interaction; ion pairsThe four Glu-Lys and the two Asp-Lys peptides studied by Baldwin (1987, 1990) have nearly the same amino acid compositions (the 16-and 17-residue peptides differ by one alanine residue) and yet show very different helix contents, both at pH 7.0 and at pH 2.5. Consequently, ion pair interactions and charge-helix dipole interactions make important contributions to helical stability. Since the initial report by Marqusee and Baldwin (1987), several other groups have demonstrated the importance of electrostatic interactions in the stability of a-helical peptides. Merutka and Stellwagen (1991) have investigated the effects of different acidic (A) and basic (B) residues on the stability of a peptide related to Marqusee and Baldwin's i + 4 AB. Gans et al. (1991) demonstrated, by using peptides comprised of only 8 Glu and

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“…There has been much progress in determining the helix propensities of residues in a variety of experimental systems (Chakrabartty et al, 1991;Lyu et al, 1990;Merutka et al, 1990;O'Neil & DeGrado, 1990; Padmanabhan et al, 1990), and a recent summary has appeared (Chakrabartty & Baldwin, 1993). The role of specific side-chain interactions in stabilizing the helical structure of a peptide has been appreciated for some time [for review, see Scholtz and Baldwin (1992)l; however, a detailed quantitation of these interactions has not been investigated to the extent necessary for a complete understanding of the problem (Armstrong & Baldwin, 1993;Gans et al, 1991;Huyghues-Despointes et al, 1993a).…”

mentioning

confidence: 99%

The energetics of ion-pair and hydrogen-bonding interactions in a helical peptide

Scholtz

Qian²,

Robbins³

et al. 1993

Biochemistry

298

326

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A single pair of Glu and Lys residues has been placed at four different spacings, and in both orientations, in an otherwise neutral alanineglutamine peptide helix, and the contribution to helix stability of the different Glu-Lys interactions has been measured. The contribution from the interaction of each charged side chain with the helix macrodipole has also been determined. A side-chain interaction between Gln and Glu, when the spacing is (i, i+4), has been detected and quantified. The interactions have been divided into contributions from hydrogen bonds (independent of the concentration of NaC1) and from electrostatic interactions (present in 10 mM NaCl, absent in 2.5 M NaCl). The major results are as follows: (1) The (i, i+3) and (i, i+4) Glu-Lys interactions are helix-stabilizing and are similar in strength to each other, regardless of the orientation of the side chains. (2) Hydrogen bonds provide the major contribution to these side-chain interactions, as shown by the following facts. First, the major part of the interaction observed in 10 m M NaCl, pH 7, is still present in 2.5 M NaCl. Second, the interaction found at pH 2 is equally as strong as that found in 2.5 M NaCl at pH 7. (3) The (i, i+4) Gln-Glu side-chain hydrogen bond is as strong as the hydrogen-bond component of the Glu-Lys interaction at both pH 2 and pH 7. The Gln-Glu interaction differs from the Glu-Lys interaction in being specific both for the orientation and the spacing of the residues. (4) No significant hydrogen-bonding interaction was found for the (i, i + l ) or (i, i+2) Glu-Lys spacings, either at pH 2 or at pH 7, in 2.5 M NaCl. At 10 mM NaCl and pH 7, these spacings show a helix-destabilizing electrostatic interaction which probably results from stabilization of the coil conformation.A complete understanding of the factors that contribute to a-helix formation by short peptides in water requires a knowledge of intrinsic helix-forming tendencies oft he separate amino acid residues as well as an accounting of the energetics of interactions between specific side chains. There has been much progress in determining the helix propensities of residues in a variety of experimental systems (Chakrabartty et al., 1991;Lyu et al., 1990;Merutka et al., 1990;O'Neil & DeGrado, 1990; Padmanabhan et al., 1990), and a recent summary has appeared (Chakrabartty & Baldwin, 1993). The role of specific side-chain interactions in stabilizing the helical structure of a peptide has been appreciated for some time [for review, see Scholtz and Baldwin (1992)l; however, a detailed quantitation of these interactions has not been investigated to the extent necessary for a complete understanding of the problem (Armstrong & Baldwin, 1993;Gans et al., 1991;Huyghues-Despointes et al., 1993a). Here we report a detailed analysis of the electrostatic interactions present in peptides with isolated Glu and Lys residues and are able to evaluate the energetics of the interaction between side chains and the interaction of a charged side chain with the helix macrodi...

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Effect of central-residue replacements on the helical stability of a monomeric peptide

Cited by 128 publications

References 22 publications

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Helical peptides with three pairs of Asp‐Arg and Glu‐Arg residues in different orientations and spacings

The energetics of ion-pair and hydrogen-bonding interactions in a helical peptide

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