Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

Fink, Anthony L.; Calciano, Linda J.; Goto, Yuji; Nishimura, M.; Swedberg, Sally A.

doi:10.1002/pro.5560020710

Cited by 67 publications

(59 citation statements)

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“…The structural properties of such intermediates are diverse, ranging from substantially unfolded to almost as structured as the native state (15). We have previously established that acid-unfolded staphylococcal nuclease (pH 2.5) can be transformed into one of three partially folded intermediates, depending on the nature and concentration of anions added to neutralize the repulsive effect from the net positive charges in the acid-unfolded polypeptide chain (16)(17). Such partially folded species have a strong propensity to aggregate: for both apomyoglobin and staphylococcal nuclease (SNase) we have observed that the propensity to associate decreased with increasing structural content, and the least structured intermediates were monomeric only at rather dilute concentrations.…”

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confidence: 99%

Association-induced folding of globular proteins

Uversky

Segel

Doniach

et al. 1998

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

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It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.Protein association (aggregation) is a major biomedical and biotechnological problem. Diseases such as the amyloidoses, prion diseases, and cataracts are caused by protein association, as are several diseases involving inclusion bodies or other amorphous deposits (e. g. inclusion body myositis, light chain deposition disease, Huntington's disease) (1, 2). Formation of inclusion bodies is a common problem in the production of recombinant proteins (3-5). The storage and delivery of protein drugs are also often complicated by the association process. Protein refolding is often also accompanied by aggregation, especially at higher protein concentrations, which has been attributed to the association of partially folded intermediates (6-11).Partially folded intermediates have been detected under both transient and equilibrium protein folding conditions (12-15). The structural properties of such intermediates are diverse, ranging from substantially unfolded to almost as structured as the native state (15). We have previously established that acid-unfolded staphylococcal nuclease (pH 2.5) can be transformed into one of three partially folded intermediates, depending on the nature and concentration of anions added to neutralize the repulsive effect from the net positive charges in the acid-unfolded polypeptide chain (16-17). Such partially folded species have a strong propensity to aggregate: for both apomyoglobin and staphylococcal nuclease (SNase) we have observed that the propensity to associate decreased with increasing structural content, and the least structured intermediates were monomeric only at rather dilute concentrations.We present here results of the effect of association on the structural properties of a partially folded intermediate of SNase. MATERIALS AND METHODSSNase. SNase was grown and purified from a cloned gene kindly supplied by D. Shortle (Johns Hopkins Univ. School of Medicine). The homogeneity of the protein samples was checked electrophoretically by using the PhastSystem (Pharmacia). Protein concentrations were determined from the published molar extinction coefficient. Salt titrations at low pH were carried out by making a series of solutions of desired salt concentration and adjusting the pH value with HCl or NaOH. Samples were incubated overnight before measurements. Sodium sulfate was from Sig...

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confidence: 99%

Association-induced folding of globular proteins

Uversky

Segel

Doniach

et al. 1998

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

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“…Other investigators (Nakano & Fink, 1990;Fink et al, 1993) have found partial structure in SNase at low pH and high salt concentrations and have called this form the "A-state.'' We believe that the SNase A-state is actually a different species from the denatured state studied herein and explore the issue further in the following paper in this issue (Carra et al, 1994).…”

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“…Many recent studies (Goto et al, 1990a(Goto et al, , 1990bAlonso et al, 1991;Buchner et al, 1991;Dryden & Weir, 1991;Fink et al, 1993) have focused on proteins under acidic conditions, following the observation at low pH of species that are often referred to as "molten globules." Intermediate forms of proteins are as yet poorly understood, but in its traditional definition (Ptitsyn, 1992) the molten globule is a compact form containing part of the secondary-structure content of the native protein but lacking specific tertiary interactions.…”

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Thermodynamics of staphylococcal nuclease denaturation. I. The acid‐denatured state

Carra¹,

Anderson²,

Privalov³

1994

Protein Science

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Using high-sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower AC,, and AH for the denaturation reaction. The acid-denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid-denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of AH on pH.

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“…Staphylococcal nuclease, a small protein (149 residues) lacking disulfide bridges, has been the subject of many early protein folding studies (reviewed in Tucker et al, 1979) and of recent renewed interest (Shortle & Meeker, 1986;Kuwajima et al, 1991;Fink et al, 1993;Green & Shortle, 1993). The crystal structures of nuclease (Hynes & Fox, 1991) and the nucleaseCa2+-3',5'-diphosphothymidine (pdTp) complex Loll & Lattman, 1989) have been refined to high resolution.…”

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Stabilization of a strained protein loop conformation through protein engineering

1995

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Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-1 17 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the 0-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "0-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of KI 16E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "0-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.Keywords: NMR; proline isomerism; protein engineering; staphylococcal nuclease In work toward rational design and redesign of protein molecules, determining the relationship between the amino acid sequence and the three-dimensional structure of protein loops and &turns is essential. These elements of protein structure comprise most of the surface of proteins (Richardson, 1981) and are often critical in protein function such as catalysis and molecular recognition (e.g., antigenhnmunoglobulin recognition; Foote & Winter, 1992). Because of their aperiodic structure, loops are the most difficult element of protein secondary structure to model. Several taxonomy schemes have been developed for the classification of &turns (Wilmot & Thornton, 1990) and more general loop conformations (Ring et al., 1992). Efforts to predict the conformation of protein loops have included comparison to similar known loop structures (Chothia et al., 1989) conformational searches utilizing Monte Carlo, simulated annealing, and exhaustive grid search techniques (Fine et al., 1986;Bruccoleri et al., 1988;Collura et al., 1993). Further work toward the understanding of the dominant interactions that determine loop conformations is important to the future of protein engineering and ration...

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Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

Cited by 67 publications

References 26 publications

Association-induced folding of globular proteins

Association-induced folding of globular proteins

Thermodynamics of staphylococcal nuclease denaturation. I. The acid‐denatured state

Stabilization of a strained protein loop conformation through protein engineering

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