Folding pathways of immunoglobulin domains. The folding kinetics of the Cgamma3 domain of human IgG1.

The in vitro folding kinetics of a fragment corresponding to an intact dimer of the Cgamma3 domain of human IgG1 (pFc') were monitored via the large changes in tryptophan fluorescence which accompany these processes. In going from the guanidine hydrochloride (Gdn.HCl) induced unfolded state (4.0 M Gdn.HCl) to the native state (0.5 M Gdn.HCl), three well-separated first-order processes were observed having time constants of 5, 50, and 350 s and roughly equal amplitudes. These values were concentration independent, a fact consistent with there being no fluorescence change accompanying dimerization. These time constants are one to two orders of magnitude slower than those observed for proteins of similar size such as ribonuclease or cytochrome c, most probably reflecting the complex processes involved in forming the correct beta-sheet arrangement of immunoglobulin domains. The corresponding unfolding transition is biphasic having time constant values of 50 and 500 s, the latter comprising 80% of the fluorescence change. These data indicate the presence of at least one species with intermediate fluorescence along the unfolding pathway. Gdn.HCl concentration jumps were also performed over various intervals within the transition zone. The results are not consistent with a fully reversible mechanism. In the absence of the intrachain disulfide bond, pFc' exists in an unfolded state even at 0.5 M Gdn.HCl. In a concomitant refolding and reoxidation experiment (at 0.5 M Gdn.HCl and using an optimal disulfide interchange catalytic system), the time constant for disulfide formation was in the range of 80--200 s and the fluorescence change revealed a lag phase analyzable in terms of rate-limiting reoxidation and refolding times consistent with those observed for the initially disulfide bonded species. Under similar conditions but a 4 M Gdn.HCl, reoxidation was more than two orders of magnitude slower, suggesting that reoxidation is directed by a refolding nucleation event.     

Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detect differential stabilization of the heme d1 and heme c moieties

The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function of guanidine hydrochloride (Gdn X HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn X HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn X HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn X HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn X HCl. The heme d1 absorbance at 638 nm decreased with increasing [Gdn X HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn X HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn X HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn X HCl, respectively. These results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.     

Viscometric characterization of pennisetin from pearl millet

Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.     

Domain characteristics of the carboxyl-terminal fragment 206–316 of thermolysin: pH and ionic strength dependence of conformation

The pH and ionic strength dependence of conformation of the COOH-terminal fragment 206–316 (fragment FII) of thermolysin was monitored by far-uv CD and difference absorption measurements. This fragment was shown previously to possess the properties of a protein domain, i.e., able to refold into a stable nativelike structure [Fontana, A., Vita, C. & Chaiken, I. M. (1983) Biopolymers 22 , 69–78]. Analysis of the CD spectra in the pH range of 1–12 indicated that near pH 1, the conformation of fragment FII appears to be in an intermediate state (H) between the fully unfolded one (U) [the guanidine hydrochloride (Gdn · HCl)-induced unfolded state] and the nativelike state (N—that attained at neutral pH). Quantitative analysis of secondary structure from CD spectra revealed that state H at 4°C is characterized by some 30% α-helical structure, compared to 47% for state N. The heat- and Gdn · HCl-mediated unfolding transitions of state H were fully reversible and characterized by little cooperativity, which is taken as an indication that state H corresponds to several species possessing different, and low, conformational stabilities. The midpoint transition from state H to N occurs near pH 2.5, implying that the acid transition results from the titration of carboxyl groups of the fragment with anomalously low pK, as would be expected for groups involved in specific salt bridges. Fragment FII at pH 1 (state H) may be induced to exhibit nearly the same degree of helicity of state N simply by increasing the ionic strength of the solution, thus reducing the repulsive interactions between positive charges within the highly charged fragment at pH 1. The results obtained emphasize the role of electrostatic interactions in the folding and stability of fragment FII and suggest a mechanism of folding of the fragment from U to N involving an intermediate state characterized by an assembly of fluctuating α-helices.     

Partially folded intermediates in insulin fibrillation

Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.     

Reduced bovine pancreatic trypsin inhibitor has a compact structure

The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions.     

Three-state protein folding: experimental determination of free-energy profile

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I<-->N and U<-->N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.     

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin

The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of helical transition of proteins by organic solvents

This paper describes a theory for the mechanism of three-state transition of proteins which is often observed in aqueous organic cosolvent systems, i.e., from the native, via intermediate to helical forms. The first transition, accompanied by changes in the tertiary and/or secondary structures, was explained by larger bindings of the organic solvent molecules to the intermediate than to the native state; the second transition, resulting in changes mainly in the secondary structure, i.e., helical transition, was explained by less hydration sites for the helical state. Computer simulations of the transition were carried out using plausible values for the number of alcohol and water binding sites of proteins as well as for the equilibrium constant of the transitions in the absence of cosolvent. A reasonable agreement with the experimental transitions was observed. The stronger effect of alcohols with longer alkyl chains was explained by their greater binding to nonpolar groups and their larger exclusion from peptide groups.     

Structural characterization of the pH-denatured states of ferricytochrome-c by synchrotron small angle X-ray scattering.

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.     

Differential scanning calorimetric and spectroscopic studies on the unfolding of Momordica charantia lectin. Similar modes of thermal and chemical denaturation

Thermal stability of Momordica charantia seed lectin (MCL) was investigated as a function of protein concentration, pH, scan rate, and at different ligand concentrations by using high-sensitivity differential scanning calorimetry (DSC). The DSC endotherm obtained at pH 7.4 consists of two entities with transition temperatures at ca. 333.7 K, and 338 K. The unfolding process is irreversible and could be described by a three-state model. For MCL tetramer ΔH_c/ΔH_v ratio is close to 4 for the first transition and ∼2 for the second transition, suggesting that four and two cooperative units are involved in the first and second transitions, respectively. In the presence of lactose both transitions shifted to higher temperatures, suggesting that ligand binds preferentially to the native conformation of MCL. Endotherms recorded as a function of pH indicate that MCL is more stable at lower pH. Chemical unfolding of MCL, induced by Gdn.HCl, was investigated by monitoring the intrinsic fluorescence properties of the protein. The results obtained indicate that chemical denaturation of MCL can also be described by a three-state process, involving an intermediate populated at ∼3–4 M Gdn.HCl. These observations suggest that the chemical and thermal unfolding processes are similar in that both of them proceed via an intermediate. The far UV and near UV CD spectra of MCL were nearly identical at different pH values and indicate that its secondary and tertiary structure do not change significantly with pH, suggesting that the structure of the protein is stable over a wide pH range.     

Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway.     

Kinetics and mechanism of the refolding of denatured ribonuclease A

On the basis of two experimental observations, it is established that the refolding mechanism of ribonuclease A (RNase A) is independent of the nature of the denaturant used [urea or guanidine hydrochloride (Gdn.HCl)]. First, by use of a double-jump technique, it is demonstrated that a similar nativelike intermediate exists on the major slow-folding pathway of both urea- and Gdn.HCl-denatured RNase A. Second, from the temperature dependence of the slow-refolding kinetics, it is shown that the activation parameters (both enthalpy and entropy) of the rate-limiting steps, as monitored by tyrosine absorbance and fluorescence, are identical for the refolding of urea- and Gdn.HCl- denatured RNase A. A refolding scheme involving one intermediate on each of the two slow-folding pathways is proposed by adopting the notion that RNase A refolds through a sequential mechanism. However, these two intermediates are formed from their respective unfolded forms (USII and USI) through two different processes of distinct physical origin. The intermediate IN, which is formed from the major slow-folding species USII through a conformational folding step, already possesses many properties of the native protein. In contrast, the intermediate (designated as I') on the minor slow-folding pathway is formed from USI by the isomerization of a proline residue (possibly Pro93) and is still conformationally unfolded. It is shown that such a refolding scheme can account for the known kinetic features of both major and minor slow-refolding pathways of RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quasi-irreversibility in the unfolding-refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride

The reversibility of the unfolding-refolding transition of horse muscle phosphoglycerate kinase, induced by guanidine hydrochloride (Gdn X HCl), was studied using the regain of enzyme activity as a probe of the native structure. An irreversibility in the reactivation process was detected when the protein was incubated in a critical concentration of denaturant (0.7 +/- 0.1 M Gdn X HCl). This apparent irreversibility was observed for the unfolding process (N----D) as well as for the refolding process (D----N). The formation of the trough followed biphasic kinetics at 23 degrees C, the first phase obeying a first-order reaction corresponded to an isomerization of an intermediate; the second phase, protein-concentration-dependent, was suppressed by lowering the temperature to 4 degrees C. The structural properties of the inactive species were studied; all the beta structures were recovered, but about 29% of the helical structures remained unfolded, and two SH groups were buried. Simulated kinetics were compared with the experimental results and were used to extend the minimum folding scheme previously proposed from equilibrium and kinetic studies [Betton et al. (1984) Biochemistry 23, 6654-6661; Betton et al. (1985) Biochemistry 24, 4570-4577]. The intermediates trapped under these conditions were structured but devoid of catalytic activity. Taking into account the structural properties of these species, the nature of the interactions involved in their formation and stabilization is discussed.     

Origins of Pressure-Induced Protein Transitions

The molecular mechanisms underlying pressure-induced protein denaturation can be analyzed based on the pressure-dependent differences in the apparent volume occupied by amino acids inside the protein and when they are exposed to water in an unfolded conformation. We present here an analysis for the peptide group and the 20 naturally occurring amino acid side chains based on volumetric parameters for the amino acids in the interior of the native state, the micelle-like interior of the pressure-induced denatured state, and the unfolded conformation modeled by N-acetyl amino acid amides. The transfer of peptide groups from the protein interior to water becomes increasingly favorable as pressure increases. Thus, solvation of peptide groups represents a major driving force in pressure-induced protein denaturation. Polar side chains do not appear to exhibit significant pressure-dependent changes in their preference for the protein interior or solvent. The transfer of nonpolar side chains from the protein interior to water becomes more unfavorable as pressure increases. We conclude that a sizeable population of nonpolar side chains remains buried inside a solvent-inaccessible core of the pressure-induced denatured state. At elevated pressures, this core may become packed almost as tightly as the interior of the native state. The presence and partial disappearance of large intraglobular voids is another driving force facilitating pressure-induced denaturation of individual proteins. Our data also have implications for the kinetics of protein folding and shed light on the nature of the folding transition state ensemble.     

Conformational properties of the unfolded state of Im7 in nondenaturing conditions

The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, ¹⁵N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R_h = 25 Å) relative to the urea-denatured state (R_h ≥ 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.     

Urea and guanidine hydrochloride induced dissociation and denaturation of bacteriophage F2 capsid.

A single, low molecular weight protein is found after urea or guanidine hydrochloride (Gdn.HCl) treatment of empty capsids derived from bacteriophage f2. The final product of denaturation is apparently a monomer, existing as a random coil in larger than or equal to 4.0 M Gdn.HCl but in a less extended form in 8.0 M urea. In contrast, an 11 S protein component is isolated after treatment of the intact virus with 4.0 M Gdn.HCl (Zelazo & Haschemeyer, 1969), indicating that RNA plays a role in stabilizing larger subunits. Denaturation by Gdn.HCl occurs in two stages as measured by changes in CD and Stokes radius: dissociation that involves a structural perturbation of aromatic side chains, followed by a major, cooperative transition that evidently results in the loss of all noncovalent structure. Denaturation by urea appears to be a much less cooperative process that occurs in several steps over a wide range of urea concentration (1--7 M). In both urea and Gdn.HCl, dissociation into subunits begins at a lower concentration of denaturant than the major changes in conformation.