Guanidine Hydrochloride Denaturation Research Articles

Role of Hydrogen Bond Networks and Dynamics in Positive and Negative Cooperative Stabilization of a Protein

Cooperativity mediated through hydrogen bond networks in yeast iso-1-cytochrome c was studied using a thermodynamic triple mutant cycle. Three known stabilizing mutations, Asn 26 to His, Asn 52 to Ile, and Tyr 67 to Phe, were used to construct the triple mutant cycle. The side chain of His 26, a wild-type residue, forms two hydrogen bonds that bridge two substructures of the wild-type protein, and Tyr 67 and Asn 52 are part of an extensive buried hydrogen bond network. The stabilities of all variants in the triple mutant cycle were determined by guanidine hydrochloride denaturation methods and used to determine the pairwise, Delta(2)G(int), and triple interaction energies. His 26 and Ile 52 interact cooperatively (Delta(2)G(int) is 1-2 kcal/mol), whereas the two other pairs of mutations interact anticooperatively (Delta(2)G(int) is -0.5 to -1.5 kcal/mol). Previously reported structural data for iso-1-cytochrome c variants containing these mutations show that changes in the strength of the His 26 to Glu 44 hydrogen bond, apparently caused by changes in main chain dynamics, provide a mechanism for the long distance (His 26 to Phe 67 and His 26 to Ile 52) propagation of pairwise interaction energies. Opposing changes in the strength of the His 26 to Glu 44 hydrogen bond caused by the N52I and Y67F mutations generate a negative triple interaction energy (-0.9 +/-0.7 kcal/mol) that combined with cancellation of cooperative and anticooperative pairwise interactions produce apparent additivity for the stabilizing effects of the single mutations in the triple mutant variant.

The Acidic C-terminal Domain Stabilizes the Chaperone Function of Protein Disulfide Isomerase

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a chaperone and catalyzes the formation and rearrangement of disulfide bonds in proteins. Domain c-(463-491), containing 18 acidic residues, is an interesting and important C-terminal extension of PDI. In this study, the PDI mutant abb'a', in which domain c is truncated, was used to investigate the relationship between the C-terminal structure and chaperone function. Reactivation and light-scattering experiments show that both wild-type PDI and abb'a' interact with lactate dehydrogenase (LDH, EC 1.1.1.27), which tends to self-aggregate during reactivation. The interaction enhances reactivation of LDH and reduces aggregation. According to these results, it seems as if domain c might be dispensable to the chaperone function of PDI. However, abb'a' is prone to self-aggregation and causes increased aggregation of LDH during thermal denaturation. In contrast, wild-type PDI remains active as a chaperone under these conditions and prevents self-aggregation of LDH. Furthermore, measurements of intrinsic fluorescence and difference absorbance during denaturation show that abb'a' is much more labile to heat or guanidine hydrochloride denaturation than wild-type PDI. This suggests that domain c is required for the stabilization and maintenance of the chaperone function of PDI under extreme conditions.

Chaperone-Like Manner of Human Neuronal Tau Towards Lactate Dehydrogenase

In our experiments, inactivation of lactate dehydrogenase (LDH, EC1.1.1.27) in the presence of human microtubule-associated tau is observably suppressed during thermal and guanidine hydrochloride (GdnHCl) denaturation. Kinetic studies show tau can prevent LDH from self-aggregation monitored by light scattering during thermal denaturation. On the other hand, neuronal tau promotes reactivation of LDH and suppresses self-aggregation of non-native LDH when GdnHCl solution is diluted. Furthermore, the reactivation yield of LDH decreases significantly with delayed addition of tau. All experiments were completed in the reducing buffer with 1 mM DTT to avoid between tau and LDH forming the covalent bonds during unfolding and refolding. Thus, Tau prevents proteins from misfolding and aggregating into insoluble, nonfunctional inclusions and assists them to refold to reach the stable native state by binding to the exposed hydrophobic patches on proteins instead of by forming or breaking covalent bonds. Additionally, tau remarkably enhances reactivation of GDH (glutamic dehydrogenase, EC 1.4.1.3), another carbohydrate metabolic enzyme, also showing a chaperone-like manner. It suggests that neuronal tau non-specifically functions a chaperone-like protein towards the enzymes of carbohydrate metabolism.

Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II.

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO(2) hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (T(m)) of 16 degrees C for the mutant compared to 55 degrees C for the wild-type protein. GuHCl-denaturation (at 4 degrees C) showed that the native state of the mutant was destabilized by 9.2kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 degrees C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9kcal/mol in accordance with its strong affinity. Acetazolamide shifts the T(m) to 34 degrees C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

Thermodynamic evaluation of a covalently bonded transition state analogue inhibitor: inhibition of beta-lactamases by phosphonates.

Serine beta-lactamases are inhibited by phosphonate monoesters in a reaction that involves phosphonylation of the active site serine residue. This reaction is much more rapid than the hydrolysis of these inhibitors in solution under the same conditions. The beta-lactamase active site therefore must have the ability to stabilize not only the anionic tetrahedral transition states of the acyl transfer reactions of substrates but also the pentacoordinated transition state(s) of phosphyl transfer reactions. A series of p-nitrophenyl arylphosphonates have been synthesized and the rate constants for their inhibition of the class C beta-lactamase of Enterobacter cloacae P99 determined. There is no direct correlation between these rate constants and the dissociation constants of analogous aryl boronic acids, where the latter are believed to generate good tetrahedral transition state analogue structures. Thus, the mode of stabilization of pentacoordinated phosphorus transition states by the beta-lactamase active site is qualitatively different from that of tetrahedral transition states. Molecular modeling suggests that the difference arises from different positioning of the side chain and of one of the oxygen ligands. In principle, the quality of the stable tetrahedral phosphonate complex as a transition state analogue structure can be assessed from the effect of its formation on the stability of the protein. Phosphonylation of the P99 beta-lactamase, however, had little effect on the stability of the protein, as measured both by thermal and guanidine hydrochloride denaturation. Consideration of the results of similar experiments with the Staphylococcus aureus PC1 beta-lactamase, where considerable stabilization is observed in thermal melting and, to a lesser degree, in formation of the molten globule in guanidine hydrochloride, but not in the complete unfolding transition in guanidine, suggests that results from the method may be strongly influenced by the interactions of the ligand with its environment in the unfolded state of the protein. Thus, quantitative estimates of the quality of a covalently bonded transition state analogue cannot generally be achieved by this method.

Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme.

The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation.

The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability

Roles of the two tryptophane residues of dimeric arginine kinase (AK) were individually investigated by site-directed mutagenesis. Both residues were fully conserved in the phosphogen kinase family and the mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, fluorescence quenching experiments, thermal stability and conformational stability. Our studies revealed that Trp-218 was located at the active site of AK and was the major fluorescence contributor (96.9%). Single replacement of this residue by alanine led to almost complete inactivation of the enzyme. In addition, a decrease in the melting temperature in differential scanning calorimetry (DSC) profiles and the equilibrium studies in guanidine hydrochloride (GdnHCl) denaturation after mutagenesis also suggested that Trp-218 takes part in stabilizing the conformational structure of AK. Although another tryptophane, Trp-208 was not located at the active sites, it may take part in maintaining the correct dimer conformation for catalysis. Replacement of this tryptophane by alanine decreased the activity to 70.3% and made it susceptible to heat and denaturants, such as GdnHCl. In addition, Trp-208 also seemed to play an important role in correct protein folding.

Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide.

Investigations of protein-solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid-dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 M). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid-inactivated porcine heart lactate dehydrogenase reflects a non-native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non-native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid-dissociated porcine heart lactate dehydrogenase as well as other viscosity-dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.

Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles.

A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c. The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.

Streptococcus pneumoniae Hyaluronate Lyase Contains Two Non-cooperative Independent Folding/Unfolding Structural Domains: CHARACTERIZATION OF FUNCTIONAL DOMAIN AND INHIBITORS OF ENZYME

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact N-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL.

Concerted Folding and Binding of a Flexible Colicin Domain to Its Periplasmic Receptor TolA

Compared with folded structures, natively unfolded protein domains are over-represented in protein-protein and protein-DNA interactions. Such domains are common features of all colicins and are required for their translocation across the outer membrane of the target Escherichia coli cell. All of these domains bind to at least one periplasmic protein of the Tol or Ton family. Similar domains are found in Ton-dependent outer membrane transporters, indicating they may interact in a related manner. In this article we have studied binding of the colicin N translocation domain to its periplasmic receptor TolA, by fluorescence resonance energy transfer (FRET) using fluorescent probes attached to engineered cysteine residues and NMR techniques. The domain exhibits a random coil circular dichroism spectrum. However, FRET revealed that guanidinium hydrochloride denaturation caused increases in all measured intramolecular distances showing that, although natively unfolded, the domain is not extended. Furthermore NMR reported a compact hydrodynamic radius of 18 A. Nevertheless the FRET-derived distances changed upon binding to TolA indicating a significant structural rearrangement. Using 1H-15N NMR we show that, when bound, the peptide switches from a disordered state to an ordered state. The kinetics of binding and the associated structural change were measured by stopped-flow methods, and both events appear to occur simultaneously. The data therefore suggest that this molecular recognition involves the concerted binding and folding of a flexible but collapsed state.

Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects.

Single site mutations that reverse or neutralize a surface charge were made at 22 ionizable residues in staphylococcal nuclease. Unfolding free energies were obtained by guanidine hydrochloride denaturation. These data, in conjunction with previously obtained stabilities of the corresponding alanine mutants, unequivocally show that the dominant contribution to stability for virtually all of the wild-type side chains examined is the electrostatic effect associated with each residue's charged group. With only a few exceptions, these charges stabilize the native state, with an average loss of 0.5 kcal/mol of stability upon neutralization of a charge. When the charge is reversed, the average destabilization is doubled. Structure-based calculations of electrostatic free energy with the continuum method based on the finite difference solution to the linearized Poisson-Boltzmann equation reproduce the observed energetics when the polarizability in the protein interior is represented with a dielectric constant of 20. However, in some cases, large differences are found, giving insight into possible areas for improvement of the calculations. In particular, it appears that the assumptions made in the calculations about the absence of electrostatic interactions in the denatured state and the energetic consequences of dynamic fluctuations in the native state will have to be further explored.

Acyl chains are responsible for the irreversibility in the Escherichia coli α-hemolysin binding to membranes

Hemolysin (HlyA) is an extracellular protein secreted by uropathogenic strains of Escherichia coli. The mature HlyA is able to bind to mammalian target cell membranes including those of the immune system, causing lysis. The lytic activity is absolutely dependent upon the Hlyc-dependent acylation of Prohemolysin. In this paper we show, through Trp fluorescence studies and denaturation in Guanidine hydrochloride, that the acylation is responsible for the loose conformation of the active protein, necessary to transform it from soluble to membrane-bound form. Previous studies showed that toxin binding to the bilayers occurs in, at least two ways, a reversible adsorption and irreversible insertion. We demonstrated that the irreversibility is due to the acyl chains in the HlyA, as shown by the protein transfer from multilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) to large unilamellar vesicles containing POPC-doxyl as protein fluorescence quencher.

Guanidine hydrochloride denaturation of glycosylated and deglycosylated stem bromelain.

Glycosylation is one of the major naturally occurring covalent modifications of proteins. We have used stem bromelain, a thiol protease with a single, N-glycosylated polypeptide chain as a model to investigate the role of glycosylation of proteins. Periodate oxidation was used to obtain the deglycosylated form of the enzyme. Denaturation studies in the presence of guanidine hydrochloride (Gn*HCl) were performed using fluorescence and circular dichroism spectroscopy. The glycosylated stem bromelain was found to be stabilized by 1.9 kcal/mol as compared to the deglycosylated one. At a given concentration of denaturant, the fraction of denatured protein was higher in the case of deglycosylated stem bromelain. In short, deglycosylated bromelain showed more susceptibility towards guanidine hydrochloride denaturation, indicating the contribution of the carbohydrate part of the glycoprotein to the stability of the enzyme.

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus

A [3Fe–4S] 1+/0 ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe–4S] 1+/0, with an unusually low reduction potential of −650 mV, determined by cyclic voltammetry at pH 7.6. [ 1H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe–4S] 1+/0 centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe–4S] 2+/1+ cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism. The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint ( T m) for thermally induced unfolding is 102±2 °C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 °C). The iron–sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.

DnaK-Sigma 32 Interaction Is Temperature-dependent: IMPLICATION FOR THE MECHANISM OF HEAT SHOCK RESPONSE

The heat shock response in bacteria is a complex phenomenon in which sigma 32 plays the central role. The DnaK/J chaperone system binds and promotes degradation of sigma 32 at lower temperatures. At heat shock temperatures, the DnaK/J-mediated degradation of sigma 32 is largely abolished by a mechanism, which is not yet fully understood. In this article we have shown that interaction of DnaK with sigma 32 is highly temperature-dependent. This interaction is completely abolished at 42 degrees C. To investigate the origin of such strong temperature dependence, we have monitored the structural changes that occur in the sigma 32 protein upon upshift of temperature and attempted to elucidate its functional roles. Upon a shift of temperature from 30 to 42 degrees C, the CD spectrum of sigma 32 becomes significantly more positive without significant change in either tryptophan fluorescence spectra or quenchability to external quenchers. 1,8-Anilinonaphthalene sulfonic acid binding at 42 degrees C is not significantly affected. The equilibrium guanidine hydrochloride denaturation of sigma 32 is biphasic. The first phase shifts to even lower guanidine hydrochloride concentrations at 42 degrees C, whereas the major phase remains largely unchanged. The sigma 32-core interaction remains unchanged as a function of temperature. This suggests that increased temperature destabilizes a structural element. We discuss the possible location of this temperature-sensitive structural element.

Screening of antagonists based on induced dissociation of a calmodulin–melittin interaction entrapped in a sol–gel derived matrix

Sol–gel derived materials offer a unique advantage for the development of sensing and screening platforms in that they allow for the entrapment of multiple species within a confined space. In this work, we show that it is possible to entrap an intact protein–peptide interaction, consisting of bovine calmodulin (bCaM) and melittin, into a sol–gel derived silicate material. Fluorescence emission data demonstrate that the entrapped complex behaves similarly to the complex in solution, and can undergo reversible dissociation upon introduction of the denaturant guanidine hydrochloride. Screening of antagonists of the bCaM–melittin complex was accomplished based on induced dissociation of the entrapped complex, which was followed by measuring the loss of sensitization of Tb(III) luminescence originating from energy transfer from the Trp of melittin to Tb(III) bound in the loops of bCaM. This study shows that entrapped protein–peptide complexes can be used as targets for drug screening or for fluorescence-based biosensing.

Role of the N-terminal helix I for dimerization and stability of the calcium-binding protein S100B.

Human S100B(beta beta) is a small intracellular EF-hand calcium-binding protein that consists of two noncovalently associated 91-residue beta monomers. The three-dimensional structures of S100B reveal the dimer interface consists of four alpha-helices (I, I' and IV, IV') packed in an X-type bundle. In this study, guanidine hydrochloride denaturation and dynamic light scattering were used to assess the impact of single (L3A, L3S, M7A, I11A, F14A) and double (L3A/I11A and L3A/F14A) substitution mutations in helix I on the stability and dimerization propensity of S100B. The free energy of unfolding (Delta G(u)) of wild-type apo-S100B was determined to be 72.4 +/- 4.0 kJ mol(-1), consistent with it being the most stable calcium-binding protein to date. The order of stability of the mutants in their apo form is S100B > L3A > L3S > I11A > M7A approximately L3A/I11A > F14A > L3A/F14A. Further, there is a strong correlation between the stability and the cooperativity of unfolding. Each mutation proved to be more stable in its calcium form compared to its apo form. The calcium-bound L3S substitution proved to be significantly more stable than calcium-saturated S100B, whereas the L3A, I11A, and L3A/I11A mutants are only slightly more stable than the wild-type protein. The F14A and L3A/F14A mutants are significantly reduced in stability, even in the presence of calcium.

Direct detection of the protein quaternary structure and denatured entity by small-angle scattering: guanidine hydrochloride denaturation of chaperonin protein GroEL

A change in the higher-order structure of an oligomeric protein is directly detectable by small-angle scattering. A small-angle X-ray scattering (SAXS) study of the denaturation process of the chaperonin protein GroEL by guanidine hydrochloride (GdnHCl) showed that the disappearance of the quaternary structure can be monitored by using a Kratky plot of the scattered intensities, demonstrating the advantage of the SAXS method over other indirect methods, such as light scattering, circular dichroism (CD), fluorescence and sedimentation. The collapse of the quaternary structure was detected at a GdnHCl concentration of 0.8 Mfor a solution containing ADP (adenosine diphosphate)/Mg2+(2 mM)/K+. From pairwise plots of the change in forward scattering intensityJ(0)/C(weight-average molecular weight) and thez-average (root mean square) radius of gyration against the GdnHCl concentration, the stability and nature of the denatured protein can be determined. The SAXS results suggest that the GroEL tetradecamer directly dissociates to the unfolded coil without going through a globular monomer state. The denatured ensemble is not a single unfolded monomer coil particle, but some mixture of entangled aggregates and a monomer of the coil molecules. Small-angle scattering is a powerful method for the detection and study of changes in quaternary and higher-order structures of oligomeric proteins.

The intrachain disulfide bond of beta(2)-microglobulin is not essential for the immunoglobulin fold at neutral pH, but is essential for amyloid fibril formation at acidic pH.

beta(2)-Microglobulin (beta2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. beta2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant beta2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced beta2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact beta2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced beta2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of beta2M.