Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein

Regan, Lynne; Rockwell, A. L.; Wasserman, Zelda R.; DeGrado, William F.

doi:10.1002/pro.5560031225

Cited by 26 publications

(22 citation statements)

References 39 publications

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“…It has been suggested that because the rn value reflects the difference in the amount of denaturant bound to the denatured versus the folded protein, differences in m values within a series of mutants may reflect differences in the solvent-accessible hydrophobic surface area that is exposed upon unfolding (Schellman, 1978). This interpretation is supported by the observation that the introduction of a disulfide bridge in a protein decreases the magnitude of the protein's m value (Villafranca et al, 1987;Pace et a!., 1988;Betz & Pielak, 1992;Regan et al, 1994). Furthermore, for several proteins the magnitude of the m value correlates with the heat capacity change that is associated with protein unfolding (ACp) (Myers et al, 1995).…”

Section: Equilibrium Guhcl Denaturation: Circular Dichroismmentioning

confidence: 99%

Surface point mutations that significantly alter the structure and stability of a protein's denatured state

Smith

Anderson

et al. 1996

Protein Science

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Significantly different m values (1.9-2.7 kcal mol-' M") were observed for point mutations at a single, solventexposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCI) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (mi-) and low ( m -) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 s" for the m+ mutant and 952 s" for the m-mutant) and the rates of unfolding (1 I s-l for m+ mutant and 3 s-I for the m-mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (at = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m f mutant did not change with increasing denaturant concentrations (Rgd -23 8,); whereas, the R8d of the m-mutant increased from approximately 17 8, to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCI-induced denatured state of these two proteins.

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Section: Equilibrium Guhcl Denaturation: Circular Dichroismmentioning

confidence: 99%

Surface point mutations that significantly alter the structure and stability of a protein's denatured state

Smith

Anderson

et al. 1996

Protein Science

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“…3 and 4). While the effect of the disulfide bridge on stability is difficult to interpret because it involves the comparison of a bimolecular concentration-dependent unfolding process for the reduced coiled-coil and a unimolecular concentration-independent unfolding process for the oxidized coiled-coil (Regan et al, 1994), we have estimated the interchain disulfide bridge to offer about 3-4 kcal/mol additional stability to the coiled-coil (Hodges et al, 1990;Kohn et al, 1995). This effect would be expected to be capable of counteracting a large number of interchain electrostatic repulsions, which as stated above have been estimated to destabilize by 0.45 kcal/mol per Glu-Glu repulsion.…”

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confidence: 99%

The Effects of Interhelical Electrostatic Repulsions between Glutamic Acid Residues in Controlling the Dimerization and Stability of Two-stranded α-Helical Coiled-coils

Kohn¹,

Monera²,

Kay³

et al. 1995

Journal of Biological Chemistry

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The effects of interhelical electrostatic repulsions in controlling the dimerization and stability of twostranded ␣-helical coiled-coils have been studied using de novo designed synthetic coiled-coils. A native coiledcoil was synthesized, which consisted of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain 2-2 disulfide bridge. This peptide, designed to contain no intrachain or interchain electrostatic interactions, forms a stable coiledcoil structure at 20°C in benign medium (50 mM KCl, 25 mM PO 4 , pH 7) with a [urea]1 ⁄2 value of 6.1 M. Five mutant coiled-coils were designed in which Gln residues at the e and g positions of the heptad repeat were substituted with Glu systematically from the N terminus toward the C terminus, resulting in each polypeptide chain having 2, 4, 6, 8, or 10 Glu residues. These substituted Glu residues are able to form interchain i to i؉5 electrostatic repulsions across the dimer interface. As the number of interchain repulsions increases, a steady loss of helical content is observed by circular dichroism spectroscopy. The effects of the interchain Glu-Glu repulsions on the coiled-coil structure are partly overcome by the presence of an interchain disulfide bridge; the peptide with six Glu substitutions is only 15% helical in the reduced form but 85% helical in the oxidized form. The stabilities of the coiled-coils were determined by urea and guanidine hydrochloride (GdnHCl) denaturation studies at 20°C. The stabilities of the coiled-coils determined by urea denaturation indicate a decrease in stability, which correlates with an increasing number of interchain repulsions ([urea]1 ⁄2 values ranging from 8.4 to 3.7 M in the presence of 3 M KCl). In contrast, all coiled-coils had similar stabilities when determined by GdnHCl denaturation (approximately 2.9 M). KCl could not effectively screen the effects of interchain repulsions on coiled-coil stability as compared to GdnHCl.

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“…This effect was demonstrated in many studies in which disulfide formation was used as a probe for structure and flexibility of proteins (for a recent work, see Ref. 17). The precomplex only needs to be short-lived because dissociation of the final complex is rendered impossible by the disulfide bridges.…”

Section: Discussionmentioning

confidence: 99%

The Function of the NC1 Domains in Type IV Collagen

Ries

Engel

Lustig

et al. 1995

Journal of Biological Chemistry

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At its C terminus, the collagen IV molecule bears a globular NC1 domain, to which two functions have been assigned. In the macromolecular network of collagen IV, two molecules are connected via their NC1 domains, which form a hexameric complex, stabilized by intermolecular disulfide bonds. In addition, the NC1 domains are thought to be responsible for chain selection and assembly. In order to understand the role of the NC1 domains during these steps, hexameric complexes were isolated and further investigated. SDS-polyacrylamide gel electrophoresis and Western blot revealed disulfidelinked ␣1(IV)NC1 and ␣2(IV)NC1 homodimers but no heterodimers. The hexamers were dissociated at low pH, separated into monomers and dimers, and submitted to reconstitution experiments. Only ␣1(IV)NC1 dimers were able to reconstitute a hexameric complex. ␣1(IV)-NC1 and ␣2(IV)NC1 monomers as well as the ␣2(IV)NC1 dimers showed only a low tendency to form complexes. It is assumed that during formation of the collagen IV network, lateral aggregation of the molecules via the triple helical domains brings the C termini of two molecules into close vicinity and that subsequently the weak interactions observed between the NC1 subdomains provide the correct alignment for a disulfide exchange. It is, however, questionable whether the low affinity between the NC1 subdomains alone is sufficient for chain assembly and alignment of the ␣(IV) chains before molecule formation.

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Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein

Cited by 26 publications

References 39 publications

Surface point mutations that significantly alter the structure and stability of a protein's denatured state

Surface point mutations that significantly alter the structure and stability of a protein's denatured state

The Effects of Interhelical Electrostatic Repulsions between Glutamic Acid Residues in Controlling the Dimerization and Stability of Two-stranded α-Helical Coiled-coils

The Function of the NC1 Domains in Type IV Collagen

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