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R H Pain

Publications and source records attributed to R H Pain.

At least 19 recordsLinked to original sources
PubMed1992

'All-or-none' mechanism of the molten globule unfolding.

The Gdm-HCl-induced unfolding of bovine carbonic anhydrase B and S. aureus beta-lactamase was studied at 4 degrees C by a variety of methods. With the use of FPLC it has been shown that within the transition from the molten globule to the unfolded state the distribution function of molecular dimensions is bimodal. This means that equilibrium intermediates between the molten globule and the unfolded states are absent, i.e. the molten globule unfolding follows the 'all-or-none' mechanism.

Carbonic Anhydrases↗
PubMed1992

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.

Acrylamide↗
PubMed1992

Intermediates in denaturation of a small globular protein, recombinant human stefin B.

Guanidinium HCl (GdmHCl), pH, and heat denaturation of the recombinant human stefin B, a low molecular weight protein inhibitor of cysteine proteinases, has been followed by circular dichroism. From the noncoincidence of the transitions in the near and far UV, the existence of stable intermediate states possessing few persistent tertiary interactions but most of the native-like secondary structure, was inferred. These intermediate states exist at equilibrium under various conditions, namely, state G at 1.7 M GdmHCl (pH 8, 25 degrees C), state A at pH 4 (0.6 M GdmHCl, 25 degrees C) and state T above 68 degrees C. By size exclusion chromatography, their apparent compactness was determined. The intermediate states A, T, and G were compact and are therefore classified as "molten globule" states.

Chromatography, Gel↗
PubMed1992

Effects of glycosylation on the folding and stability of human, recombinant and cleaved alpha 1-antitrypsin.

The equilibrium unfolding transitions for the human M form of alpha 1-antitrypsin have been determined using a number of techniques reflecting changes in tryptophan fluorescence lifetime and quenching, exposure of tryptophan to solvent, secondary structure and the Stokes' radius of the protein. The denaturation curves are more complex than is usual for globular proteins and indicate the presence of multiple equilibrium intermediates in the presence of denaturant. This is in marked contrast to the more co-operative transition of the cleaved inhibitor. In addition, a recombinant non-glycosylated alpha 1-antitrypsin has been shown to have a closely similar conformation to the human M protein and to exhibit very similar reversible unfolding transitions, and hence similar stability and co-operativity. Differences in tryptophan environment are reflected in the dequenching of tryptophan fluorescence and reduced asymmetry in the near ultraviolet circular dichroism of the non-glycosylated protein, suggesting direct interaction of glycosyl residues with a tryptophan. Both the M type and the recombinant protein exhibit similar patterns of folding, with rapid collapse to a compact intermediate reminiscent of the widely observed molten globule state that folds more slowly to the native protein. The papain-cleaved M form also folds through a similar compact state in the absence of the C-terminal peptide that results from cleavage. It is concluded that part of the C-terminal 36 residue peptide interacts strongly with the main body of the protein in the folded inhibitor. This interaction will also be important during early stages of folding of the intact protein to direct the folding pathway. The lack of glycosylation leads to an increase in aggregation of the recombinant protein upon refolding, especially after extended denaturation times. The more rapid turnover of the recombinant protein in vivo is shown not to be due to a lower thermodynamic stability, but may be associated with a lower kinetic stability arising from the increased tendency to aggregation.

Chromatography, Gel↗
PubMed1992

Denaturation of stefin B by GuHCl, pH and heat; evidence for molten globule intermediates.

GuHCl, pH and thermal denaturation of the recombinant stefin B was followed by circular dichroism (CD) and size-exclusion chromatography (SEC). CD at 277 nm was taken as an indicator of integral tertiary structure and CD at 222 nm as an indicator of the secondary structure. Compactness was expressed by the volumes of elution on a SEC (Superose 12) column. Data on equilibrium denaturation were recalculated to the fractions of the native state (fN). The results have shown that equilibrium intermediates of the molten globule type exist under conditions of low pH, high temperature or medium GuHCl concentrations, namely A, T and G. Recent findings on structure and energetics of molten globule intermediates are reviewed.

Circular Dichroism↗
PubMed1991

Refolding and assembly of penicillin acylase, an enzyme composed of two polypeptide chains that result from proteolytic activation.

The in vitro folding and assembly of penicillin acylase (EC 3.5.1.11) (PA) to active enzyme has been studied. PA is a large bacterial protein (Mr = 86,000) comprising two peptides, alpha and beta, produced by proteolytic processing and activation of a 92-kDa precursor. Proteins that result from proteolytic processing are characteristically difficult if not impossible to refold. Different factors that affect folding and assembly of PA, including pH, ionic strength, and temperature, have been studied. Yields of 60% can be obtained, based on recovery of enzyme activity, together with another 20% of folded and associated monomer with conformation closely similar to that of the active enzyme but with the active site not formed. Evidence is presented for in vitro assembly proceeding via initial folding of the N-terminal alpha-peptide with subsequent collapse of the transiently folded beta-chain on to the surface of the former. A slow process of rearrangement follows association in vitro. Competition experiments support the proposal that the linker endopeptide in the precursor serves to increase the probability of productive collision between folded alpha- and beta-peptides. The effect of raised temperature is to interfere with the folding of the alpha-peptide, thus preventing proper folding of the precursor. This finding accounts for the basis of the temperature regulation of PA production in vivo.

Enzyme Activation↗
PubMed1991

Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus.

3-Phosphoglycerate kinases from yeast and the extreme thermophilic bacterium Thermus thermophilus HB8 have been used as models for investigating the relationship between stability, dynamics and activity. It was found that while at a given temperature the thermophilic protein is more stable, its conformational dynamics as measured by the ability of acrylamide to quench the fluorescence of a buried tryptophan as well as its specific activity, are both lower than for the mesophilic protein. As the temperature is increased, the thermodynamic stability of the thermophilic protein approaches that of the mesophilic protein at its working temperature. Its conformational dynamics and specific activity however were both shown to increase, until at the physiologically operational temperature, they become similar to those of the mesophilic enzyme at its operational temperature. These results confirm the proposal that a direct relationship and balance holds between thermodynamic stability, dynamics and specific activity in globular proteins. They demonstrate also the constraining effect of increased stability upon conformational dynamics and enzyme activity.

Acrylamide↗
PubMed1991

Resolution of the fluorescence of the buried tryptophan in yeast 3-phosphoglycerate kinase using succinimide.

The heterogeneous fluorescence of yeast 3-phosphoglycerate kinase, a hinge-bending enzyme with two tryptophans, has been resolved into two approximately equal components, one accessible and one inaccessible to the relatively inefficient quencher succinimide. The inaccessible component is blue-shifted and exhibits a heterogeneous fluorescence decay which has a temperature-dependence and steady-state acrylamide quenching properties typical of a single tryptophan in a buried environment. This component is therefore assigned to the buried tryptophan W333. The presence of succinimide greatly simplifies the fluorescence allowing the conformational dynamics of the buried tryptophan and its environment to be studied without interference from the other tryptophan.

Calorimetry↗
PubMed1991

Molten globule intermediates and protein folding.

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.

Animals↗
PubMed1990

The folding and solution conformation of penicillin G acylase.

The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by near- and far-ultraviolet circular dichroism, steady-state and time-resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid-point of 4.5 M urea. Separation of its constituent alpha and beta peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated beta peptide aggregates over a wide range of urea concentrations but that the alpha peptide refolds reversibly to a compact state. Physical studies showed that the refolded alpha peptide has a compact but asymmetric structure with more alpha helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8-anilino-1-naphthalene sulfonic acid binding and which is normally covered by the beta peptide in the native enzyme. The results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.

Amidohydrolases↗
PubMed1990

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

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 alpha-lact-albumin, bovine beta-lactoglobulin, yeast phosphoglycerate kinase, beta-lactamase from Staphylococcus aureus and recombinant human interleukin 1 beta, 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 (beta, alpha + beta and alpha/beta), 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.

Fluorescence↗
PubMed1989

Assignment of the heterogeneous static and time-resolved tryptophan fluorescence of 3-phosphoglycerate kinase.

The heterogeneous fluorescence of yeast 3-phosphoglycerate kinase, a hinge-bending enzyme with two tryptophan residues, has been resolved into three emission components using steady-state and time-resolved studies of the fluorescence quenching by acrylamide, iodide and caesium ions at different emission wavelengths. The buried Trp-333 has a blue-shifted heterogeneous emission spectrum characterised by three fluorescence lifetimes, and is inaccessible to quenchers. The surface Trp-308 also has a heterogeneous emission with multiple lifetimes. The emission of Trp-308 can be separated into a blue-shifted emission accessible to acrylamide and caesium only, and a red-shifted emission accessible to all three quenchers.

Kinetics↗
PubMed1989

Determination of the contributions of individual aromatic residues to the CD spectrum of IL-1 beta using site directed mutagenesis.

Circular dichroism (CD) spectra have been measured in the aromatic region for recombinant human interleukin IL-1 beta and for site specific mutants in which each of the four tyrosines and the single tryptophan residue have been replaced one at a time by phenylalanine. These mutant proteins have been shown to have conformations that are closely similar to that of the wild type protein. By comparing the spectrum of each mutant with that of the wild type human protein it has been possible to assess the contribution of each aromatic residue to the CD spectrum of the latter. It has been shown that the spectrum is largely a result of contributions from Trp 120 and from Tyr 68.

Circular Dichroism↗
PubMed1988

The 'molten globule' state is involved in the translocation of proteins across membranes?

Strong evidence exists that the translocation of proteins across a variety of membranes involves a non-native or denatured conformational states. On the other hand a compact state having secondary but not rigid tertiary structure and called the 'molten globule' state has been identified as being stable under mild denaturing conditions. A similar state has been shown to accumulate on the folding pathway of globular proteins. These states are compact though sufficiently expanded to include water, and they are internally mobile. It is proposed that these molten globule states may be suitable candidates for protein translocation across biological membranes.

Biological Transport↗