Polyproline fold—In imparting kinetic stability to an alkaline serine endopeptidase

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0 M concentration, organic solvents viz. methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymotrypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1 M GdnHCl upto 8 h, in organic solvents (75% v/v) for 72 h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60 °C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein.     

Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies

Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.     

Artocarpus hirsuta lectin. Differential modes of chemical and thermal denaturation.

Unfolding, inactivation and dissociation of the lectin from Artocarpus hirsuta seeds were studied by chemical (guanidine hydrochloride, GdnHCl) and thermal denaturation. Conformational transitions were monitored by intrinsic fluorescence and circular dichroism. The gradual red shift in the emission maxima of the native protein from 335 to 356 nm, change in the ellipticity at 218 nm and simultaneous decrease in the sugar binding activity were observed with increasing concentration of GdnHCl in the pH range between 4.0 and 9.0. The unfolding and inactivation by GdnHCl were partially reversible. Gel filtration of the lectin in presence of 1-6 m GdnHCl showed that the protein dissociates reversibly into partially unfolded dimer and then irreversibly into unfolded inactive monomer. Thermal denaturation was irreversible. The lectin loses activity rapidly above 45 degrees C. The exposure of hydrophobic patches, distorted secondary structure and formation of insoluble aggregates of the thermally inactivated protein probably leads to the irreversible denaturation.     

Synthesis and conformational analysis of salivary proline‐rich peptide P‐B

The 57‐amino acid human salivary polypeptide P‐B has been synthesized by the solid‐phase method using 9‐fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier‐transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P‐B. Examination of the CD spectra of P‐B showed the content of the secondary structure to be independent of temperature over the range 0–60 °C at pH = 7 as well as over the pH range of 2–12 at 37 °C. P‐B adopts predominantly unordered structure with locally appearing β‐turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type‐II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C‐terminal fragment of the peptide (Pro⁵¹–Pro⁵⁴), which constitutes 7%. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.     

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Multistate unfolding of α-mannosidase from Canavalia ensiformis (Jack Bean): evidence for the thermostable molten globule

The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II α-mannosidase from Canavalia ensiformis (Jack Bean) (Jbα-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jbα-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jbα-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 °C and stability of the structure up to 80 °C unlike the native Jack Bean α-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II α-mannosidase.     

Structural stability and unfolding transition of β-glucosidases: a comparative investigation on isozymes from a thermo-tolerant yeast

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C _m, occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T _m, (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0–3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0–4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k _unf of native protein to be 5.407 × 10^?5 s^?1 and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.     

Quantitative Determination of Site-Specific Conformational Distributions in an Unfolded Protein by Solid-State Nuclear Magnetic Resonance

Solid-state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid-state ¹³C NMR spectra of samples prepared with uniform ¹³C labeling of selected residues show relatively sharp cross-peaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most cross-peaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid-state NMR techniques that provide independent constraints on backbone ? and ψ torsion angles in samples with sequential pairs of carbonyl ¹³C labels. Without GdnHCl, the combined data are well fit by α-helical conformations. At [GdnHCl] = 4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of α-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl] = 7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid-state NMR.     

GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ～8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.     

Detection of an equilibrium intermediate in the folding of a monomeric insulin analog.

To determine the conformational properties of the C-terminal region of the insulin B-chain relative to the helical core of the molecule, we have investigated the fluorescence properties of an insulin analog in which amino acids B28 and B29 have been substituted with a tryptophan and proline residue respectively, ([WB28,PB29]insulin). The biological properties and far-UV circular dichroism (CD) spectrum of the molecule indicate that the conformation is similar to that of native human insulin. Guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation of the analog as monitored by CD intensity at 224 nm indicates a single cooperative transition with a midpoint of 4.9 M GdnHCl. In contrast, when the equilibrium denaturation is observed by steady-state fluorescence emission intensity at 350 nm, two distinct transitions are observed. The first transition accounts for 60% of the observed signal and has a midpoint of 1.5 M GdnHCl. The second transition roughly parallels that observed by CD measurements with an approximate midpoint of 4.5 M GdnHCl. The near-UV CD spectrum, size-exclusion, and ultracentrifugation properties of [WB28,PB29]insulin indicate that this analog does not self-associate in a concentration-dependent manner as does human insulin. Thus, the observed fluorescence changes must be due to specific conformational transitions which occur upon unfolding of the insulin monomer with the product of the first transition representing a stable folding intermediate of this molecule.     

Bromophenol blue binding as a probe to study urea and guanidine hydrochloride denaturation of bovine serum albumin

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525-675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference (DeltaAbs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, DeltaGDH2O as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.     

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

Mechanism of Unfolding of Goat Lung Cystatin During Urea and Guanidine Hydrochloride Induced Denaturation

Cystatins essentially regulate lysosomal cysteine protease besides affecting several physiological processes. In the present study, denaturation of a high molecular weight cystatin (Mr 66.4 kDa) purified from goat lung (GLC-I) has been studied by monitoring its inhibitory activity, intrinsic fluorescence, circular dichroism (CD), and binding of ANS. It was found that increasing concentration of GdnHCl significantly enhances the inactivation and unfolding of the purified inhibitor (GLC-I) with complete loss of inhibitory activity at 4 M GdnHCl. Denaturation of GLC-I in the presence of GdnHCl is accompanied by red shift (15 nm) of the emission maximum as shown by intrinsic fluorescence. The inhibitory activity of GLC-I was increased by 1.5 fold at 2 M urea; however, it decreased with further increased of the urea concentration. Intrinsic fluorescence studies of GLC-I in the presence of 0–3 M urea shows blue shift of 5 nm, suggesting stabilization of the inhibitor followed by 5 nm red shift at higher concentration. ANS binding studies in the presence of urea indicate significant changes in the tertiary structure of the inhibitor. Thus, our result shows denaturation profile of GLC-I following simple two state transitions in the presence of GdnHCl while it proceeds through an intermediate state in the presence of urea.     

Protein unfolding studies of thiol-proteinase inhibitor from goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) muscle in the presence of urea and GdnHCl as denaturants

In recent years, many advances have been made in the understanding of functional and structural characteristics of protein evolution from denaturant-based studies that subject the protein to a change in the microenvironment. This paper reports the chemical denaturation of purified goat muscle cystatin (GMC) a thiol-proteinase inhibitor, using urea and guanidine hydrochloride (GdnHCl). The subtle conformational changes of GMC were monitored by intrinsic fluorescence, extrinsic fluorescence, and CD spectroscopic techniques. Further, the activity of GMC as a function of increasing concentration of denaturants was also studied. It was found that increasing the concentration of GdnHCl significantly enhances the inactivation and unfolding of the inhibitor (GMC). In urea-induced denaturation, the intrinsic and extrinsic fluorescence intensity reveals significant structural changes in the inhibitor. Further, it was found that at low concentrations of urea, up to 0.5–1.0 M, there was quenching of fluorescence intensity compared with the native form and a red shift of 5 nm was observed up to 5–8 M. The results presented in this paper suggest that GdnHCl-induced denaturation of GMC follows a simple two-state rule in which native → denatured state transition occurs in a single step. However denaturation with urea proceeds through an intermediate or non-native state.     

Autophosphorylation dependent destabilization of the insulin receptor kinase domain: tryptophan-1175 reports changes in the catalytic cleft

Protein kinases are regulated by conformational or chemical changes which facilitate access of substrates to the active site and promote correct orientations of catalytically essential residues and water molecules. The switch between basal and activated states of the insulin receptor's kinase domain (IRKD) results from autophosphorylation. We investigated the effects of IRKD autophosphorylation on the conformational stability by guanidine hydrochloride (GdnHCl) dependent denaturation and by iodide quenching of intrinsic fluorescence. Tryptophan residues of the recombinant soluble IRKD (residues R953-S1355) were excited at a lambdaex of 295 nm, and emission spectra were analyzed for centroid (a characteristic of average polarity of the indole rings' environments) and integrated fluorescence intensity over the lambdaem range of 310-420 nm. Denaturation profiles of both apo- and phospho-IRKD forms are complex with at least three distinct unfolding transitions. The first and last transitions were reversible and cooperative and had midpoints at 0.4 or 0.7 M GdnHCl and 2.4 or 2.7 M GdnHCl, respectively; transitions of phospho-IRKD occurred at lower GdnHCl concentrations. Calculations of free energy of unfolding suggested a loss of approximately 2.3 kcal/mol of stabilization for the first transition and approximately 1.5 kcal/mol for the third transition. Circular dichroism showed subtle changes in secondary structure over the first transition and global unfolding over the last transition. The first transition reports changes primarily in the local environment of W1175, which is near the catalytic loop and is conserved among protein tyrosine kinases. W1175 is also the dominant fluorophore of the native emission spectrum. Iodide quenching of W1175 was virtually undetectable in the apo-IRKD but significant in the phospho-IRKD, suggesting that W1175 exposure to small solutes is strongly dependent on the conformation of the activation loop. These studies indicate that autophosphorylation, while exposing the catalytic center, also produces a conformer less stable than the apoenzyme.