Side Chain Contributions to the Stability of Alpha-Helical Structure in Peptides

Lyu, Ping‐Chiang; Liff, Mark I.; Marky, Luis A.; Kallenbach, Neville R.

doi:10.1126/science.2237416

Cited by 487 publications

(408 citation statements)

References 39 publications

(5 reference statements)

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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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“…We have designed a heterodimeric coiledcoil ( Figure 1 and Table 1), based on principles revealed by more than 15 years of investigation (13,16,17,22). The primary stabilizing interactions are the hydrophobic interactions in the core (34,37), electrostatic attractions across the interface (5,7,9,16), and helical propensity (16,(38)(39)(40)(41). Valine and leucine were chosen for positions a and d, as they are known to form a highly stable hydrophobic core (35,36,42,43).…”

Section: Resultsmentioning

confidence: 99%

“…Although serine has a low helical propensity, it is the smallest polar, uncharged residue that can increase solubility while minimally interacting with the other side-chains. To compensate, position c was occupied with alanine, the amino acid residue with the highest helical propensity (16,(38)(39)(40)(41). Although nominally a hydrophobe, alanine has the smallest side-chain, which should discourage aggregation.…”

Section: Resultsmentioning

confidence: 99%

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...

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“…The side-chain rotamer conformations of the straight-chain Leu, Abu, Nval, or Nle are less restricted in an a-helix than the p-branched Val and Ile or the aromatic Tyr or Phe (MacGregor et al, 1987). The loss in sidechain conformational entropy when both interacting residues are straight-chain up to the Cy carbon (Leu-Leu, Abu-Abu, NvalNval, or Nle-Nle) would be considerable if they were fixed in Restriction of side chain conformations helps to rationalize the lower helix propensities of Val, Ile, Phe, and Tyr relative to Leu, Abu, Nval, or Nle observed experimentally (Lyu et al, 1990;O'Neil & DeGrado, 1990;Padmanabhan et al, 1990;Park et al, 1993;Chakrabartty et al, 1994) and verified by Monte Carlo calculations (Creamer & Rose, 1992). Our present results indicate that it also plays an important role in determining the strength of interactions between nonpolar side chains in a helix.…”

Section: Peptide Design and CD Propertiesmentioning

confidence: 99%

Tests for helix‐stabilizing interactions between various nonpolar side chains in alanine‐based peptides

Padmanabhan

Baldwin

1994

Protein Science

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Straight-chain, non-natural, nonpolar amino acids norleucine, norvaline, and a-amino-n-butyric acid at various spacings do not interact with themselves to stabilize helix formation in alanine-based peptides, but do interact with a Tyr spaced i, i + 4 to stabilize alanine helices, similar to the helix-stabilizing i, i + 4 Tyr-Leu and Tyr-Val interactions reported earlier (Padmanabhan s, Baldwin RL, 1994, JMol Bioi241 :706-713). Leu spaced i, i + 4 from another Leu is measurably helix-stabilizing relative to the corresponding i , i + 3 pair, but less so than for i, i + 4Val-Leu, Ile-Leu, or Phe-Leu pairs (relative to the corresponding i, i + 3 pairs) when Leu is C-terminal to the other nonpolar amino acid. Our results indicate that limited side-chain flexibility in an a-helix strongly favors the interaction between 2 nonpolar residues to stabilize an isolated a-helix.

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Side Chain Contributions to the Stability of Alpha-Helical Structure in Peptides

Cited by 487 publications

References 39 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

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Tests for helix‐stabilizing interactions between various nonpolar side chains in alanine‐based peptides

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