Reversible Denaturation Research Articles

Properties of Hybrid Active Sites in Oligomeric Proteins: Kinetic and Ligand Binding Studies with Chloramphenicol Acetyltransferase Trimers

Alteration of the charge of surface lysyl residues of chloramphenicol acetyltransferase (CAT) by site-directed mutagenesis was used to increase the charge difference between the subunits of two naturally occurring enzyme variants (CATI and CATIII). The introduced charge change greatly facilitates the purification of CATI/CATIII and CATIII/CATIII hybrid trimers by ion-exchange chromatography. Hybrids containing only one functional active site per trimer were generated in vitro by reversible denaturation of mixtures of "active" subunits (retention of a catalytic histidine at position 195) and "inactive" subunits (with alanine replacing histidine 195). Such hybrids were used (1) to demonstrate that the previously observed novel binding of a steroidal antibiotic (fusidic acid) by CATI involves amino acid residues at each subunit interface and (2) to identify specific residues contributing to such interactions. A pre-steady-state kinetic characterization of homotrimers containing the H195A substitution also revealed that fusidate binding to CATI may, like chloramphenicol binding, involve a hydrogen bond with the catalytic histidine residue. In addition, confirmation of the fact that His-195 interacts with chloramphenicol in CATI as well as in CATIII makes it likely that it is essential for the catalytic mechanism of all naturally occurring variants of CAT, as first suggested by structural evidence for the type III enzyme (Leslie, 1990).

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.

The temperature-induced denaturation of small globular proteins as a first-order phase transition of ‘crystal molecules’

A general feature of temperature-induced reversible denaturation of small globular proteins is its all-or-none character. This strong cooperativity leads to think that protein molecules, possessing only two accessible thermodynamic states, the native and the denatured one, resemble ‘crystal molecules’ that melt at raising temperature. An analysis, grounded on mean field theory, allows to conclude that the two-state transition is a first-order phase transition. The implication of this conclusion are briefly discussed.

Cold denaturation of an icosahedral virus. The role of entropy in virus assembly.

Assembly of icosahedral viruses is not completely understood at the molecular level. The main puzzle is to answer how chemically identical protein subunits take up unique positionally dependent conformations during the process of assembly. The stability of the ribonucleoprotein particles of cowpea mosaic virus (CPMV) to pressures and subzero temperatures has been studied. At room temperature, reversible pressure denaturation of CPMV is obtained only in the presence of 5.0 M urea. On the other hand, when the temperature is decreased to -15 degrees C, the ribonucleoprotein components denature, at 2.5 kbar, in the presence of 1.0 M urea. At temperatures close to -20 degrees C, denaturation is obtained even in the absence of urea. Whereas the denaturation promoted by pressure and urea at room temperature is reversible, virus particles denatured when the temperature is decreased under pressure cannot reassemble. Bis-ANS binding data suggest that this irreversibility may be related to protein release from RNA, which probably does not occur under denaturating conditions at room temperature. The contributions of enthalpy (delta H*) and entropy (delta S*) for the free energy of association of CPMV are calculated from the cold denaturation curves under pressure. The entropy change is positive and large, making the assembly of ribonucleoprotein components an entropy-driven process, suggesting that the burial of nonpolar side chains during the process of assembly is the structural foundation for CPMV assembly.

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease were investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8 kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change.

Inhibitor Binding in the Transition State for Unfolding of Adenosine Deaminase

Inhibitors protect calf intestinal adenosine deaminase from reversible denaturation by guanidine hydrochloride, and from heat inactivation. In the presence of saturating concentrations of ligands, the first-order rate constant for reversible denaturation of this enzyme in 3.0 M guanidine hydrochloride is reduced 6-fold by pteridine or 6-dimethylaminopurine ribonucleoside, and 60-fold by purine ribonucleoside; these compounds provide similar protection against thermal inactivation at 70°C. These protective effects indicate that in the transition state for denaturation by heat or guanidine hydrochloride, the enzyme retains much of its native structure, exhibiting 60-70% of the free energy of association with these ligands that was present in the native enzyme in the ground state. However, neither these ligands nor the extremely powerful inhibitor 2′-deoxycoformycin affect the rate of refolding of enzyme denatured in guanidine hydrochloride.

A Thermodynamic Scale for the .beta.-Sheet Forming Tendencies of the Amino Acids

The results of a study to measure the beta-sheet forming propensities of the 20 naturally occurring amino acids are presented. The protein host for these studies is the 56 amino acid B1 domain of staphylococcal IgG binding protein G [Fahnestock, S.R., Alexander, P., Nagle, J., & Filpula, D. (1986) J. Bacteriol. 167, 870-880]. This protein was selected because it exhibits a reversible two-state thermal denaturation transition and its structure is known at high resolution. A suitable guest position in the protein was identified, and its neighboring environment was modified to minimize the potential for artifactual interactions. All 20 amino acids were individually substituted at the guest site, and their effect on the protein's thermal stability was determined. NMR was used to verify the structural integrity of several of the proteins with different amino acid substitutions at the guest site. The results of these studies provide a thermodynamic scale for the relative beta-sheet forming propensities of the amino acids that shows a clear correlation with the beta-sheet preferences derived from statistical surveys of proteins of known structure.

Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH

The reversible acid denaturation of tumour necrosis factor, TNFα, a trimeric, all-β protein, leads to significant conformational changes within the molecule. A change in far UV CD spectra reveals a shift in the secondary structure content of the protein, with α-helical structure being induced. Loss of ellipticity in the near UV reflects a loss of tertiary interactions. This form of TNF is both compact and trimeric, as revealed by fluorescence anisotropy and sedimentation velocity analysis, respectively. Acid-denatured TNF therefore possesses the defining features of the molten globule intermediate while retaining the ability of the still incompletely folded monomers to exhibit the surface specificity necessary for maintaining the trimeric state.

Detection of intermediate protein conformations by room temperature tryptophan phosphorescence spectroscopy during denaturation of Escherichia coli alkaline phosphatase

The reversible denaturation of Escherichia coli alkaline phosphatase (AP) was followed by monitoring changes in enzymatic activity as well as by measurements of the time-resolved room temperature phosphorescence from Trp 109. It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCI) inactivate AP by different mechanisms as reflected by differences in the time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate. Time-resolved tryptophan phosphorescence is exquisitely sensitive to changes in the local environment of the emitting residue, unlike the steady state phosphorescence intensity which is a composite of both the lifetime and concentration of the emitting protein species. The results show that while inactivation in EDTA proceeds by loss of the zinc ion as expected, denaturation in acid or GdnHCl produces a heterogeneous population of AP molecules, detected by a distribution analysis of the phosphorescence lifetime, which may reflect multiple pathways to the final unfolded state. Time-resolved phosphorescence also demonstrates the existence of an enzymatically active but structurally less rigid intermediate state during unfolding. As the rigidity decreases, the susceptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration. The experiments provide new insight into the mechanism of denaturation of AP and demonstrate the sensitivity of time-resolved room temperature phosphorescence to the structural details of intermediate states produced during unfolding of proteins.

Circular permutation of T4 lysozyme.

To examine the relationship between polypeptide chain synthesis and protein folding, we have constructed a circularly permuted variant of phage T4 lysozyme. The permuted protein begins at residue 37 of the wild-type sequence and ends at residue 36. The normal chain termini are joined by a six-residue linker, Ser-Gly4-Ala. The permuted lysozyme folds efficiently and cleaves bacterial cell walls with normal specific activity. As judged by circular dichroism, UV absorbance, fluorescence, and nuclear magnetic resonance spectroscopy, the permutation causes little change in the structure of the protein. Reversible denaturation experiments show that the permutation reduces the stability of T4 lysozyme only 0.8-1.1 kcal/mol. These results demonstrate that a protein with two domains can be permuted with little change in activity, structure, and stability. The order of chain synthesis, the sequential arrangement of secondary structures, and the position of chain termini with respect to domain boundaries do not determine the protein fold.

Correction: Guanidine Hydrochloride Stabilization of a Partially Unfolded Intermediate During the Reversible Denaturation of Protein Disulfide Isomerase

Correction: Guanidine Hydrochloride Stabilization of a Partially Unfolded Intermediate During the Reversible Denaturation of Protein Disulfide Isomerase

Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase

Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase

Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein.

Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes. The mechanism of CRP regulation most likely involves communication between subunits and domains. A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities. Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions. Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP. The denaturation of CRP was induced by guanidine hydrochloride. The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism. The spectroscopic data implied that CRP unfolds in a single cooperative transition. Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant. Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered. The functional and other structural properties of renatured and native CRP have also been examined. Quantitatively identical results were obtained. Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native<-->2(CRP)Native<-->2(CRP)Denatured. The delta G values for dissociation (delta Gd) and unfolding (delta G(u)) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 +/- 0.6 and 7.2 +/- 0.1 kcal/mol, respectively. To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively. Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible. Both S-CRP and CH-CRP exist as stable dimers with similar delta Gd values of 10.1 +/- 0.4 and 9.5 +/- 0.4 kcal/mol, respectively. Results from this study in conjunction with crystallographic data [McKay, D. B., Weber, I. T., & Stietz, T. A. (1982) J. Biol. Chem. 257, 9518-9524] indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization.(ABSTRACT TRUNCATED AT 400 WORDS)

Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase.

The reversible denaturation of protein disulfide isomerase proceeds through intermediates that are stabilized by interaction with guanidine hydrochloride. At pH 7.5, the equilibrium denaturation by urea is completely reversible and the transition can be reasonably well-described by a two-state model involving only native and denatured forms. In comparison, the equilibrium denaturation by guanidine hydrochloride occurs in two distinct steps. In the presence of a low constant amount of guanidine hydrochloride (0.5-1.4 M), urea denaturation also becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by specific interaction with guanidine hydrochloride but not by high concentrations of other salts or other denaturants.

Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH(2), their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramatically enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its "regular" counterpart.

Molten globule state of food proteins

The occurrence of the molten globule state, a unique state with a partially folded conformation that can be distinguished from either the native or the fully denatured forms, has been demonstrated in many globular proteins. The molten globule state is formed as a kinetic intermediate for reversible denaturation, in equilibrium under certain mild denaturing conditions, and upon insertion into a cell membrane during cellular translocation. Several food proteins from animal sources take on the molten globule state, which may be involved in the functional properties of these food proteins.

Soluble and immobilized enzymes in water-miscible organic solvents: glucoamylase and invertase

The efficiency of enzymes in organic solvents is determined by the catalytic parameters, which mostly are changed in comparison with those in aqueous solutions, as well as by reversible and irreversible denaturation of the enzyme protein. The interplay of these processes was studied on soluble and immobilized glucoamylase and invertase in mixtures of buffer and several water-miscible solvents such as methanol, glycerol, dimethylformamide, and dioxane. A comparison of the catalytic activity in 50 or 30% (v/v) solvents with the extent of irreversible inactivation during the time of assay showed that the solvents act on the catalytic parameters of the enzymes and their conformational stability in different ways. Irreversible inactivation is especially strong in dimethylformamide, whereas glycerol does not induce denaturation but strongly reduces the catalytic efficiency. Striking effects were observed in dioxane, where high stability to irreversible denaturation but strong reduction of catalytic power were observed. In all solvents K m values were more affected than V max values, suggesting that reversible denaturation plays an inferior role in activity losses. Similar effects of the individual solvents were also observed after immobilization of the enzymes by covalent binding to a macroporous aminomethyl polystyrene-divinylbenzene copolymer or a macroporous aminopropyl silica. In all cases, however, immobilized enzymes showed better long-term stability than the soluble enzymes.

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-field nuclear magnetic resonance relaxation study of thermal effects on milk proteins

SummaryA recently described nuclear magnetic resonance (NMR) method was evaluated for its usefulness in studying thermal effects on milk proteins. The increase in water proton T2 relaxation rate observed during thermal treatment of aqueous whey protein solutions above the denaturing onset temperature paralleled results obtained with the standard Rowland (1938) method. The influence of milk constituants on NMR characteristics was analysed. The NMR response increased with the ionic strength and the addition of caseinate or casein micelles. The relevance of the T2 relaxation probe for studying thermal modifications of milk proteins is discussed. It is proposed to apply the NMR method for determining either reversible or irreversible thermal denaturation of whey proteins in model Systems.

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.