Abstract
An antiparallel coiled-coil has been designed and characterized as a model for studying protein folding and assembly. This heterostranded antiparallel coiled-coil was formed by an interchain disulfide bond between cysteine residues at position 2 of one chain and at position 33 of the other chain. Each peptide chain has 35 residues which are composed of five heptad repeats of the sequence K-L-E-A-L-E-G with a single Leu-->Ala substitution at position 16. Two homostranded parallel coiled-coils were also formed as co-products of the oxidation reaction to form the interchain disulfide bond. The CD spectra of the parallel and antiparallel peptides were very similar and their high molar ellipticities at 220 nm did not increase in the presence of 50% trifluoroethanol. These data suggest that, like the parallel peptides, the antiparallel peptide also exists in a coiled-coil structure. Urea and guanidine hydrochloride denaturation studies, in conjunction with molecular modeling studies, suggest that there are no physical restrictions to the packing of hydrophobic residues in an antiparallel coiled-coil. However, interchain electrostatic interactions can have positive or negative contributions to the overall stability of the disulfide-bridged coiled-coil. In addition, interchain electrostatic interactions appear to play a major role in protein folding by controlling the parallel or antiparallel alignment of the alpha-helical polypeptide chains. This study is also for the first time providing us with a new understanding of the information that can be obtained from urea and guanidine hydrochloride denaturation studies of proteins concerning the contributions of hydrophobic and electrostatic interactions on stability.
Highlights
An antiparallel coiled-coil has been designed and studies of tropomyosin have contributed to our understanding characterized as a model for studying protein folding of the coiled-coil structure
In addition,interchainelectrostaticinteractionsappearto play a major role in protein folding by controlling the parallel or antiparallel alignmenoft the a-helicalpolypeptide chains
Some globular proteins have the characteristic 3-4 hydrophobic repeat and have coiled-coil domains (O’Shea et al, 1989a,1991; Banner et al, 1987; Landschulz et al, 1988; Cusack et al, 1990; Gentz et al, 1989; De Francesco et al, 1991; Hu et al, 1990; Chakerian et al, 1991;Reddy et al, providing us with a new understanding of the infor- 1992).these coiled-coilmotifs seem to be associated mation that can be obtained from urea and guanidine with a very specific function, as a result of dimerization, as hydrochloride denaturation studies of proteins con- opposed to just being part of the overall folded structure of cerning the contributionsof hydrophobic and electro- the protein
Summary
In the presence of 50%, TFE the [O]2zo/[0]20ra8tio decreased to 0.91, which is characteristic of single-stranded or non-interacting a-helices These data, taken together, indicate that these oxidized peptides, including the C2A16/C33A16 antimatogram of the products after 16 h of stirring the heterostranded peptide (C2A16/C33A16 ox, 1 mg/ml) in a redox buffer (250 p~ oxidized glutathione, 250 pM reduced glutathione, 0.2 M KCl, 0.1 M Tris, 1mM EDTA, pH 8.7) (O’Shea et al, 1989a, 1989b). The [GdnHC1]l/zvalues (Table I), obtained by non-linear curve-fitting program of the GdnHCl denaturation data (Fig. 4A),showed that the antiparallel coiled-coilwas more stable stranded peptide was formed These results indicatethat the formation of the heterostranded antiparallel peptide was not than the parallel coiled-coils The [Den a t ~ r a n t ] v~a/l~uesfor the parallel and antiparallel coiledcoils crossed-over in the 35-50% GdnHCl region
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