The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Cis proline mutants of ribonuclease A. II. Elimination of the slow-folding forms by mutation.

Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanidine Hydrochloride Unfolding of a Carbonic Anhydrase Molten Globule

Guanidine hydrochloride-induced unfolding of a carbonic anhydrase molten globule was studied by high-resolution nuclear magnetic resonance spectroscopy. The study resulted in estimation of the number of water and denaturant molecules bound to the molten globule at various denaturant concentrations in solution. When compared with the data on unfolding of native carbonic anhydrase, these estimates indicate that the unfolding is underlain by an increased local concentration of the denaturant near the protein molecule, which results from the increased ratio between guanidine hydrochloride-bound and protein-bound waters.     

Protein folding: Hypotheses and experiments

The current state of the problem of protein folding is reviewed with special attention to the novel molten globule state of the protein molecule, intermediate between the native and unfolded states. Experimental evidence on the existence of this state and its role in protein folding are compared with the sequential model of protein folding proposed by the author in 1972–1973.     

Heterogeneous Packing in the Folding/Unfolding Intermediate State of Bitter Gourd Trypsin Inhibitor

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2–3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.     

Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM -ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (NIU) followed by fluorescence at 363 nm, (NI) by fluorescence at 318 nm, and CD (IU) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5–6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.     

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.     

Models for protein folding and nature's choice of protein as catalyst

The study of protein folding and unfolding pathways lends a fascinating dimension to protein biochemistry. Several models for protein folding have been postulated. Two powerful probes used in protein folding study are far UV-CD monitored stopped flow kinetics and pulse hydrogen exchange in conjunction with NMR. The formation of molten globule, which is an intermediate possessing secondary structure but not a well packed tertiary structure, is now emerging as a common feature on the folding pathway of many proteins. The molten globule is recognized by a class of molecules called chaperones which act as accelerators of protein folding. This article ends by elucidating why proteins are Nature's choice as catalysts.     

Folding of bovine growth hormone is consistent with the molten globule hypothesis

Previous results from equilibrium and kinetic studies of the folding of bovine growth hormone (bGH) have demonstrated that bGH does not follow a simple two-step folding mechanism. These results are summarized and interpreted according to the "molten globule" model. The molten globule state of bGH is characterized as a folding intermediate which is largely alpha-helical, retains a compact hydrodynamic radius, has packing of the aromatic side chains that is similar to the unfolded state, and possesses a solvent-exposed hydrophobic surface along helix 106-127 that readily leads to association.     

Structural characterization of MG and pre-MG states of proteins by MD simulations,NMR, and other techniques

Almost all proteins fold via a number of partially structured intermediates such as molten globule (MG) and pre-molten globule states. Understanding the structure of these intermediates at atomic level is often a challenge, as these states are observed under extreme conditions of pH, temperature, and chemical denaturants. Furthermore, several other processes such as chemical modification, site-directed mutagenesis (or point mutation), and cleavage of covalent bond of natural proteins often lead to MG like partially unfolded conformation. However, the dynamic nature of proteins in these states makes them unsuitable for most structure determination at atomic level. Intermediate states studied so far have been characterized mostly by circular dichroism, fluorescence, viscosity, dynamic light scattering measurements, dye binding, infrared techniques, molecular dynamics simulations, etc. There is a limited amount of structural data available on these intermediate states by nuclear magnetic resonance (NMR) and hence there is a need to characterize these states at the molecular level. In this review, we present characterization of equilibrium intermediates by biophysical techniques with special reference to NMR.     

Local and global cooperativity in the human alpha-lactalbumin molten globule

NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P, the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of alpha-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions.     

Partially Folded Conformations of Inositol Monophosphatase Endowed with Catalytic Activity

The stability of porcine brain inositol monophosphatase in the presence of increasing concentrations of urea was investigated at pH 7.5. Exposure of the enzyme to 8 M urea brings about the dissociation of the dimeric species of 58 kDa into monomeric forms as revealed by gel filtration chromatography. Unfolding of the protein by 8 M urea results in a decrease of the ellipticity at 220 nm (20%) together with a perturbation of the near-UV circular dichroism spectrum. Urea-treated inositol monophosphatase binds Co²⁺ ions with a dissociation constant of 3.3 M. The enzyme is catalytically competent when assayed with 4-nitrophenyl-phosphate in the presence of the activating ion Co²⁺ at pH 7.5 in 8 M urea. The apparent activation constant for Co²⁺ is 2.5 mM. It is postulated that partially folded conformations of monomeric species preserve their catalytic function because the affinity of Co²⁺ ions for the metal coordination center of the protein is not perturbed by exposure to 8 M urea.