Conformation of a stable intermediate on the folding pathway of Staphylococcus aureus penicillinase

Carrey, Elizabeth A.; Pain, Roger H.

doi:10.1016/0005-2795(78)90542-1

Cited by 62 publications

(44 citation statements)

References 30 publications

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“…The multistate behavior was attributed to weak intersubunit interactions that give rise to monomeric folding intermediates (Carrey & Pain, 1978;Zetina & Goldberg, 1980). In other cases, greater separation between the native-intermediate and the intermediate-unfolded transitions leads to an inflection in the transition curve, e.g., X repressor (Banik et al, 1992) and aspartate aminotransferase (Herold & Kirschner, 1990).…”

Section: Discussionmentioning

confidence: 99%

Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models

1993

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Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wildtype aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.Keywords: cooperativity; folding intermediate; folding mechanism; protein stability; tryptophan fluorescence; tryptophan mutations Fluorescence spectroscopy is a sensitive and potentially informative method of monitoring both equilibrium and kinetic properties of protein-folding reactions. The hydrophobic character of aromatic residues (i.e., tryptophan, tyrosine, and phenylalanine) often leads to their being sequestered in the interior of the native conformation. Therefore, the intensity, emission maximum, and distribution of various lifetime components of these residues can differ from those exposed to solvent (Beechem & Brand, 1985). Disruption of noncovalent interactions that define the native form and exposure of these aromatic side chains to solvent during a global unfolding reaction can lead to changes that are observed readily in these static and dynamic properties (see, for example, the accompanying paper by Royer et al. [1993] A challenge for kinetic studies of folding reactions has been to obtain specific assignments and structural interpretations for observed changes in fluorescence properties. Garvey et al. (1989) solved this problem in dihydrofolate reductase from Escherichia coli by using site-directed mutagenesis to replace several of the tryptophan residues individually. These workers found that one of the kinetic phases detected in the refolding of wild-type reductase was eliminated selectively in the mutant, in which Trp 74 was replaced by Phe. This effect was attributed to the burying of Trp 74 in a hydrophobic cluster early in the folding process. Later studies demonstrated that Trp 74 packs against Trp 47 in a native-like fashion in a transient folding intermediate (Kuwajima et al., 1991).With this precedent, a set of mutations in the tryptophan aporepressor from E. coli were constructed with the goal of determining the assignments of the three kinetic phases detected previously by tryptophan fluorescence during refolding of this dimeric protein (Gittelman

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Section: Discussionmentioning

confidence: 99%

Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models

1993

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“…For /Mactamase state H (neutral pH, 2 M urea), for example, the far UV ellipticity has an intensity 85% that of the native state (Carrey and Pain 1978). In the cases of ~-lactalbumin (Baum et al 1989) and cytochrome c (Roder et al 1988), both at low pH, the use of NMR has shown that the helices that are present are located at positions in the sequence closely similar to those in the native state.…”

Section: Stable States Of Intermediate Conformationmentioning

confidence: 99%

“…Different proteins exhibit stable intermediate states whose equivalent hydrodynamic radii of gyration or Stokes radii range from approximately one to 2 times those of the respective native states. Where all other properties coincide with those for a compact or molten globule state, it is proposed that those proteins with larger volumes, e.g./Mactamase (H state) (Carrey and Pain 1978) and apomyoglobin (A state) (Goto et al 1990 a) are mainly compact states that include the secondary structure, with extended random coil loops or chain termini.…”

Section: The State Is Largely Globularmentioning

confidence: 99%

Molten globule intermediates and protein folding

1991

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The background to the concept of the term "molten globule" as a description of intermediates observed in the folding of globular proteins is discussed. These compact intermediates are characterised by certain properties including the presence of secondary structure and considerable conformational mobility compared to the native, functional state. Those intermediates that are thermodynamically stable under mild denaturing conditions have many features in common with the transient intermediates that accumulate significantly during the process of folding. Attention is drawn to cases where the two types are however distinguished on grounds of their Stokes radius, in which cases there is currently no direct evidence for the involvement of the stable intermediates on the folding pathway. Experimental evidence relating to the early stages in folding is reviewed and compared, highlighting the temporal relationship between general collapse of the polypeptide chain and the formation of secondary structure. The continued use of the term "molten globule" is recommended where the minimum essential structural criteria for this state are met.

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“…The remainder of the native ellipticity is regained with the same rate constants as for near UV c.d., activity and native elution volume. The results obtained by stoppedflow techniques for ribonuclease [ 161,lysozyme [17], cytochrome c [20], ,8LG [20] and parvalbumin [21] show that secondary structure is formed early in protein folding before N structure formation. These results demonstrate that a substantial amount of N-like, secondary structure is present in the compact state I.…”

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confidence: 99%

Evidence for a molten globule state as a general intermediate in protein folding

et al. 1990

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The folding of globular proteins occurs through intermediate states whose characterisation provides information about the mechanism of folding. A major class of intermediate,states is the compact 'molten globule', whose characteristics have been studied intensively in those conditions in which it is stable (at acid pH, high temperatures and intermediate concentrations of strong denaturants). In studies involving bovine carbonic anhydrase, human a-lact-albumin, bovine /3-lactoglobulin, yeast phosphoglycerate kinase, D-lactamase from Staphylococcus aurew and recombinant human interleukin l/I, we have demonstrated that a transient intermediate which accumulates during refolding is compact and has the properties of the 'molten globule' state. We show that it is formed within 0.1-0.2 s. These proteins belong to different structural types (8, a+/3 and a/S), with and without disulphide bridges and they include proteins with quite different times of complete folding (from seconds to decades of minutes).We propose that the formation of the transient molten globule state occurs early on the pathway of folding of all globular proteins. Protein folding; Folding intermediate; Folding kinetics; Framework modelThe stable molten globule states obtained under mild denaturing conditions have been shown to be consistently much more compact than state U by viscosity, sedimentation, diffuse X-ray scattering, quasielastic light scattering and urea gradient electrophoresis for CAB [4], ,8Lase [5,6] and aLA [1,2,7]. It is almost as compact as state N [l-7] and has a pronounced secondary structure [l-4,6,8].This secondary structure can be N-like and the molten globule may have some features of the N fold [9]. However, this state differs from state N by the absence of close packing throughout the molecule and by a substantial increase of fluctuations in side chains as well as of larger parts of the molecule [l-3]. In agreement with these data, the equilibrium molten globule states for PLase [lo] and CAB both have Ves on FPLC gel exclusion that are intermediate between the Ves for N and U states. This permits the use of FPLC not only for the evaluation of the compactness of these states but also to monitor the kinetics of the formation of state N in refolding experiments [lo].

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Conformation of a stable intermediate on the folding pathway of Staphylococcus aureus penicillinase

Cited by 62 publications

References 30 publications

Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models

Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models

Molten globule intermediates and protein folding

Evidence for a molten globule state as a general intermediate in protein folding

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