Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein Toward Thermal Inactivation

Perry, Linda L.; Wetzel, Ronald

doi:10.1126/science.6387910

Cited by 318 publications

(154 citation statements)

References 35 publications

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“…Proteins can be engineered to be more stable in vitro with few mutations, by introduction of specific bonds (39,40) or favorable interactions (41)(42)(43). However, it has been shown that in vitro protein stabilization based on local stabilization of a secondary structural element can strengthen not only the final F state but also a small set of discrete partially folded native-like intermediates (41).…”

Section: Discussionmentioning

confidence: 99%

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Viguera

Serrano

2003

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

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Amide hydrogen͞deuterium exchange rates have been determined for two mutants of ␣-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from highenergy barrier crossings. Comparison of ⌬G ex and mex parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long ␤-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.kinetics ͉ ␤-hairpin T he Src homology 3 (SH3) domain from ␣-spectrin is a wellcharacterized model system for protein folding and stability (1). It was shown early that this domain folds by a two-state transition (2) and that its isolated secondary structure elements are quite unstructured (3). We have investigated (4) the consequences of fusing the SH3 domain with a short sequence KITVNGKTYE (BHH) that tends to fold as a stable ␤-hairpin (5). In the SHHBergerac mutant (SHH for short) the two-residue distal ␤-turn (N47-D48) of ␣-spectrin SH3 has been substituted by BHH. SHH is more stable and folds significantly faster than the short WT ␣-spectrin SH3 at moderate urea concentrations, but unlike WT, its folding rate levels off under strong folding conditions (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org), thus deviating from the simplest two-state folding mechanism. Mutation of two residues in the inserted BHH region of SHH (Ile-2Ј and Val-4Ј to Ala, SHA-Bergerac) destabilizes the protein and switches it back to a two-state folding process (Fig. 6). When fused to WT, the inserted BHH and BHH I2ЈA V4ЈA sequences adopt a fully folded ␤-hairpin structure protruding from the protein (4), but the corresponding isolated peptides are, respectively, Ϸ30% folded (BHH) and fully unfolded (BHH I2ЈA V4ЈA).Understanding the reasons behind rate constant deviations could be important in improving the methods available for in vitro protein stabilization, as well as in revealing limiting steps related to switches from two to higher order folding mechanisms. Deviations in chevron plots do not have an unequivocal interpretation. Rate constants reflect free energy differences from reactants to the highest energy level in the reaction coordinate or transition state ( ‡). The deviations often occurring at lower denaturant concentrations in refolding experiments are usually assigned to changes within the denatured ensemble, i.e., a new energy level or intermediate state (I) is stabilized...

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Section: Discussionmentioning

confidence: 99%

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Viguera

Serrano

2003

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

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“…Of these, the most extensively studied is T4 lysozyme, which has no disulfides in its native form. Five different novel disulfide bonds have been introduced into this protein (Perry & Wetzel, 1984;Wetzel et al, 1988;Matsumura et al, 1989a,b). Two have been found to lower T,,, by 20 "C at neutral pH.…”

Section: Novel Disulfidesmentioning

confidence: 99%

Disulfide bonds and the stability of globular proteins

1993

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An understanding of the forces that contribute to stability is pivotal in solving the protein-folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native-state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native-state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect. Keywords: disulfide bonds; protein stability; thermodynamicsThe determination of the forces that govern protein stability is of fundamental importance for our ability to understand and control the interactions of complex biological molecules. It has been known since the 1960s that the primary structure of a protein dictates its threedimensional fold (see Anfinsen, 1973), yet a comprehensive understanding of the factors that impart thermodynamic stability to proteins is elusive. This is because the tertiary folds of native proteins are defined by a large Reprint requests to: Stephen F. Betz, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328.Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; C,, the concentration of GdmCl at which half the protein is denatured; D-state, the denatured state of a protein; GdmCI, guanidinium chloride; hew, hen egg white; mden, the slope of the line of -AGd versus denaturant concentration; N-state, the native state of a protein; RNase, ribonuclease; Tm, temperature at which half the protein is denatured; t l I z , time required for enzymatic activity to decrease 50%; AC,, the difference in heat capacity between the denatured and native states; AGd, AG for protein denaturation; AGd,HZO, AGd determined from chemical denaturation; AGd, T, AGd determined from thermal denaturation; A H , , , the change in conformational enthalpy between the native and denatured states; N H , , AH for protein denaturation; AH&,,, the change in solvational enthalpy between the native and denatured states; AH,,,, AH for protein denaturation at T,,,; AS,,,, the change in conformational entropy between the native and denatured states; AS,, AS for protein denaturation; Ashyd, the change in solvational entropy between the native and denatured states.

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“…Increases in the conformational stability of a protein-desirable in many contexts (2)-can be realized by enhancing the hydrophobic effect (3, 4), introducing or shielding a hydrogen bond (5) or electrostatic interaction (6)(7)(8), or introducing a disulfide crosslink (9)(10)(11) or metal ion-binding site (12). These strategies are often frustrated by enthalpy-entropy compensation, whereby enthalpic stabilization is offset entirely by entropic destabilization, or vice versa (13).…”

mentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

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

153

142

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Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in ðProProGlyÞ 7 collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T m values that are increased by >50°C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 Å reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2∕3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.collagen triple helix | nonnatural amino acid | preorganization | protein stability | x-ray crystallography T he three-dimensional structures of proteins are stable because of a delicate balance of forces (1). Increases in the conformational stability of a protein-desirable in many contexts (2)-can be realized by enhancing the hydrophobic effect (3, 4), introducing or shielding a hydrogen bond (5) or electrostatic interaction (6-8), or introducing a disulfide crosslink (9-11) or metal ion-binding site (12). These strategies are often frustrated by enthalpy-entropy compensation, whereby enthalpic stabilization is offset entirely by entropic destabilization, or vice versa (13). Moreover, the strategies often lack subtlety and can lead to a misshapen and hence dysfunctional protein.As elaborated by Cram (14), the principle of preorganization states that, "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes." In a typical manifestation, a conformational constraint is imposed upon a receptor or ligand during its chemical synthesis. This principle can also yield biopolymers with increased conformational stability (Fig. 1). For example, the stability of a DNA duplex correlates with the helicity of its single strands (15, 16), and a bicyclic ("locked") derivative of ribose enhances that stability (17, 18). The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21-23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24-27).We suspected that alterations to proline residues could provide a particularly incisive means to preorganize the conformation of a polypeptide chain. In classic work, Matthews and coworkers substituted proline, which is the least flexible residu...

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Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein Toward Thermal Inactivation

Cited by 318 publications

References 35 publications

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Disulfide bonds and the stability of globular proteins

Stereoelectronic and steric effects in side chains preorganize a protein main chain

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