Refolding rate of stability‐enhanced cytochrome <i>c</i> is independent of thermodynamic driving force

McGee, William A.; Nall, Barry T.

doi:10.1002/pro.5560070501

Cited by 11 publications

(5 citation statements)

References 41 publications

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“…The most frequently isolated group of second site suppressors were global suppressors at positions 163, 166, and 170 of the coat protein (1). Global suppressors have been identified in other proteins (43)(44)(45). For example, the global suppressors of the tsf mutants in P22 tailspike protein have been shown to alleviate folding defects by decreasing the rate of aggregation through stabilization of a folding intermediate (17, 46 -48).…”

Section: Resultsmentioning

confidence: 99%

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Aramli

Teschke

2001

Journal of Biological Chemistry

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Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.The primary amino acid sequence of a polypeptide encodes all of the information necessary for folding and assembly pathways, as well as the native three-dimensional structure (2). Substitutions and deletions in the amino acid sequence of a protein can have a significant impact on the ability of a protein to fold or assemble properly. Depending on the protein, such changes in the amino acid sequence can lead to protein misfolding, mislocalization caused by misfolding, or aggregation (3, 4). Amino acid substitutions in p53 lead to a misfolding problem compromising the function of the protein, resulting in cancer (5). Further, there are the diseases that result from mislocalization caused by protein misfolding. For example, in ␣ 1 -antitrypsin deficiency, a single amino acid substitution results in the misfolding of ␣ 1 -antitrypsin, leading to the accumulation of long chain polymers within the hepatocyte. This leads to a reduction of the plasma concentrations of ␣-antitrypsin and predisposes individuals to emphysema and liver disease (6). Therefore, understanding the nature of protein grammar is of paramount interest.Although the process of protein folding is still not completely understood, it is known that larger proteins often have identifiable folding intermediates. These folding intermediates may interact inappropriately before reaching the native state. In fact, protein misfolding is a common problem faced by biotechnology companies that harvest proteins of commercial interest using recombinant DNA technologies in heterologous hosts (7-11). Often these proteins have problems with inclusion body formation, thereby decreasing the yield of pharmaceutically important products. Single amino acid substitutions can affect the folding pathway by shifting the folding from the productive pathway to off-pathway aggregation. For example, the amino acid substitutions in transthyretin causes a shift in the equilibrium between the native state and an aggregation-prone unfolding intermediate, resulting in amyloid formation. Individuals with any of the 50 known amino acid substitutions in transthyretin are predisposed to familial amyloidosis (12-15). During the folding of P22 tailspike proteins with tsf (te...

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Section: Resultsmentioning

confidence: 99%

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Aramli

Teschke

2001

Journal of Biological Chemistry

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“…Based on this observation, it has been predicted that residues involved in the folding nuclei of proteins may be conserved across naturally occurring homologous families, or exhibit intra-family conservation across superfamilies, including those of CI-2 (Shaknovich et al, 1996), cytochrome c (Ptitsyn, 1998), ubiquitin (Michnick & Shakhnovich, 1998), CheY (Mirny et al, 1998), ADAh2 (Mirny & Shakhnovich, 1999) and CD2 (Mirny & Shakhnovich, 1999). Only extremely limited mutagenesis/kinetics studies (<10 % of positions characterized) have been reported for cytochrome c (Colo'n et al, 1996;McGee & Nall, 1998), ubiquitin (Khorasanizadeh et al, 1996) or CD2 (Lorch et al, 1999), thus the conservation of kinetically signi®cant residues in these proteins is dif®cult to address at this time. As indicated above, however, we observe no statistically signi®cant evidence in support of the suggestion that residues forming the folding nucleus of CI-2 are highly conserved (Shaknovich et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Evolutionary conservation in protein folding kinetics

Plaxco

Larson

Ruczinski

et al. 2000

Journal of Molecular Biology

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“…Measured values of DG ‡ 0T are calculated from "measured" k f or k u using Equation 3~Materials and methods!. Nall, 1997;McGee & Nall, 1998!, suggesting that an analogous submillisecond phase should be observed by temperature jumps of iso-2. There are no faster phases observed by 287 nm absorbance.…”

Section: Burst Phases and Two-state Models Of Foldingmentioning

confidence: 99%

Cytochrome c folds through a smooth funnel

Panda

Benavides-Garcia²,

Pierce³

et al. 2000

Protein Science

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A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump~T-jump! relaxation technique has been used to compare the unfolding0folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time t obs is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near T m , the midpoint of the thermal unfolding transitions, t obs is four to eight times faster for H33N,H39K iso-2~t obs ; 4-10 ms! than for iso-2~t obs ; 20-30 ms!. T-jumps show that there are no kinetically unresolved~t Ͻ 1-3 ms T-jump dead time! "burst" phases for either protein. Using a two-state model, the folding~k f ! and unfolding~k u ! rate constants and the thermodynamic activation parametersare evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k obs~ϭ obs Ϫ1 ! ϭ k f ϩ k u . The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.Keywords: cytochrome c; fast folding; folding landscape; heme ligands; His; iso-2; kinetic traps; nonnative; temperature jump Recently, there have been a number of publications on the "fast folding" of small globular proteins in the submillisecond time range~Schindler et al

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Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force

Cited by 11 publications

References 41 publications

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Alleviation of a Defect in Protein Folding by Increasing the Rate of Subunit Assembly

Evolutionary conservation in protein folding kinetics

Cytochrome c folds through a smooth funnel

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