Protein stability curves

Becktel, Wayne J.; Schellman, John A.

doi:10.1002/bip.360261104

Cited by 1,053 publications

(1,025 citation statements)

References 29 publications

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“…AH is the enthalpy of unfolding at the T, of the mutant. AAG, the difference between the free energy of unfolding of the mutant and wild-type proteins, was estimated at 44°C using a thermodynamic model (Brandts & Hunt, 1967;Becktel & Schellman, 1987), which includes a constant change in heat capacity, AC,. estimated in this case to be 1.8 kcal/mol deg.…”

Section: Resultsmentioning

confidence: 99%

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

et al. 1998

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To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts 5 0.3 A); in other cases mainchain atoms moved up to 2 A.In the case of Ile 29 -+ Ala the structure col!apsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 4 Ala (V149A) and Met 6 -+ Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the. cavity. The Val 149 -+ Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 -+ Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu + Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol" k 3 . Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu + Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).

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Section: Resultsmentioning

confidence: 99%

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

et al. 1998

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“…ATm is the difference between the melting temperature of the mutant and that of wild-type lysozyme (40.75 " C ) . AAG, the difference between the free energy of unfolding of the mutant and wild-type proteins, was estimated using a thermodynamic model (Brandts & Hunt, 1967;Becktel & Schellman, 1987), which includes a constant change in heat capacity, AC,, estimated in this case to be 2.4 kcal/mol-deg. For the mutants whose melting temperatures are within a few degrees of wild type, the estimated error in AAG is kO.1 kcal/mol.…”

Section: Resultsmentioning

confidence: 99%

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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“…The dependence of AGND on T for a given set of solution conditions (pH, ionic strength, etc.) defines the stability curve for a protein (Becktel & Schellman, 1987).…”

Section: Thermodynamic Origins Of Hyperthermostabilitymentioning

confidence: 99%

X‐ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

et al. 1992

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The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 °C, have been determined by X‐ray crystallography to a resolution of 1.8 å. Crystals of this rubredoxin grow in space group P212121 with room temperature cell dimensions a = 34.6 å, b = 35.5 å, and c = 44.4 å. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X‐PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 å and 2.06°, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 å and in bond angles of 1.95°. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen‐bonding network in the β‐sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino‐terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.

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Protein stability curves

Cited by 1,053 publications

References 29 publications

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

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

X‐ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

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