High Concentrations Of GdnHCl Research Articles

Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.

Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labeling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the β-sheet have acquired structure that is protective against HX. The major difference is, however, that β2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence, the sequence of structural events is different on the two alternative pathways.

Mechanism of Human Apohemoglobin Unfolding.

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1β1 interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α1γ1 interface is more resistant to dissociation than the α1β1 interface.

A Distinct Proof on Interplay between Trehalose and Guanidinium Chloride for the Stability of Stem Bromelain.

Guanidinium chloride (GdnHCl), a potential denaturant, is well-known to denature a number of proteins in vitro as well as in vivo studies. Its deleterious action on stem bromelain (BM) is quite prominent resulting decrease in protein structure and stability. The counteraction of this adverse effect of GdnHCl by the use of osmolytes is scarcely studied and the mechanism is still illusive and not exclusive. For the first time, to test elegant and simple counteraction hypothesis as a general mechanism we utilized fluorescence, circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering to study the counteraction of GdnHCl-induced denaturation of BM by the trehalose. It is revealed from the investigation of the results that trehalose is efficiently counteracting GdnHCl undesirable impacts on BM stability at molar ratio 1:1 of trehalose and GdnHCl. On the contrary, proteolytic activity of BM is increased only for the counteraction study of BM at very high concentrations of GdnHCl although still less than BM in buffer. The mutual exclusion of both trehalose and GdnHCl may stand for the counteraction of denaturation of BM resulting in a compact conformation with less solvent exposed surface area and increased secondary and tertiary structures. In addition, a decrease in BM-solvent interactions may also be contributing to some extent as there is little binding of trehalose replacing some water molecules and reducing binding of GdnHCl.

Influence of chemical denaturants on the activity, fold and zinc status of anthrax lethal factor

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase which, through a process facilitated by protective antigen, translocates to the host cell cytosol in a partially unfolded state. In the current report, the influence of urea and guanidine hydrochloride (GdnHCl) on LF׳s catalytic function, fold and metal binding was assessed at neutral pH. Both urea and GdnHCl were found to inhibit LF prior to the onset of unfolding, with the inhibition by the latter denaturant being a consequence of its ionic strength. With the exception of demetallated LF (apoLF) in urea, unfolding, as monitored by tryptophan fluorescence spectroscopy, was found to follow a two-state (native to unfolded) mechanism. Analysis of the metal status of LF with 4-(2-pyridylazoresorcinol) (PAR) following urea or GdnHCl exposure suggests the enzyme to be capable of maintaining its metal ion passed the observed unfolding transition in a chelator-inaccessible form. Although an increase in the concentration of the denaturants eventually allowed the chelator access to the protein׳s zinc ion, such process is not correlated with the release of the metal ion. Indeed, significant dissociation of the zinc ion from LF was not observed even at 6M urea, and only high concentrations of GdnHCl (>3M) were capable of inducing the release of the metal ion from the protein. Hence, the current study demonstrates not only the propensity of LF to tightly bind its zinc ion beyond the spectroscopically determined unfolding transition, but also the utility of PAR as a structural probe.

Partial unfolding of a monoclonal antibody: role of a single domain in driving protein aggregation.

We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 °C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's CH2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the CH2 domains leads to aggregation.

Preliminary studies on the renaturation of denatured catfish (Clarias gariepinus) glutathione transferase

Purified juvenile catfish (Clarias gariepinus) glutathione transferase (cgGST) was denatured in vitro and renatured in the absence and presence of different concentrations of endogenous or xenobiotic model substrates. Protein transitions during unfolding and refolding were monitored by activity measurement as well as changes in protein conformation using UV difference spectra at 230nm. Gdn-HCl at 0.22M caused 50% inactivation of the enzyme and at 1.1M, the enzyme was completely unfolded. Refolding of cgGST main isozyme was not completely reversible at higher concentrations of Gdn-HCl and is dependent on protein concentration. An enzyme concentration of 30μg/ml yielded 40% percentage residual activity in the presence of glutathione (GSH), regardless of the concentration that was present as opposed to 30% obtained in its absence. The xenobiotic model substrate, lindane, appears to have no effect on the refolding of the enzyme. In summary, our results show that GSH assists in the refolding of cgGST in a concentration-independent manner and may be involved in the same function in vivo whereas the xenobiotic model substrate does not.

Protein-Ligand Interactions of the D-Galactose/D-Glucose-Binding Protein as a Potential Sensing Probe of Glucose Biosensors

In this paper we have studied peculiarities of protein-ligand interaction under different conditions. We have shown that guanidine hydrochloride (GdnHCI) unfolding-refolding of GGBP in the presence of glucose (Glc) is reversible, but the equilibrium curves of complex refolding-unfolding have been attained only after 10-day incubation of GGBP/Glc in the presence of GdnHCl. This effect has not been revealed at heat-induced GGBP/Glc denaturation. Slow equilibration between the native protein in GGBP/Glc complex and the unfolded state of protein in the GdnHCl presence is connected with increased viscosity of solution at moderate and high GdnHCl concentrations which interferes with diffusion of glucose molecules. Thus, the limiting step of the unfolding-refolding process of the complex GGBP/Glc is the disruption/tuning of the configuration fit between the protein in the native state and the ligand.

Beta-Sheet Features in the Greek Key Gamma D-Crystallin Recognized by the Lens Chaperone Alpha B-Crystallin

The γ-crystallins are long-lived proteins of the vertebrate lens consisting of four Greek-key motifs organized into two homologous domains. Human γD-Crys can refold in vitro to its native state after unfolding in high concentrations of GdnHCl. However, in buffer or at very low denaturant concentrations aggregation of refolding HγD-Crys intermediates competes with productive refolding. α-crystallin, a small heat shock protein chaperone, is a polydisperse complex of ∼ 800 kD consisting of homologous ∼20 kD αA- and αB-crystallin chains (αA- and αB-Crys). Its chaperone activity involves suppressing aggregation by binding aggregation-prone species. Aggregates isolated from mature-onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of βγ-crystallins. We had previously determined that the conformation of the bound HγD-Crys substrate in γD_αB complexes resembles the partially folded intermediate populated during refolding/unfolding equilibrium experiments. The chaperone/substrate interaction have been probed further using single domain constructs. The HγD-Crys C-terminal domain construct (γD-Ctd) aggregated upon refolding, while the N-terminal domain construct (γD-Ntd) did not aggregate significantly under similar conditions. HαB-Crys suppressed the aggregation of the γD-Ctd and formed long-lived γD-Ctd_αB complexes. Using a double mutant of HαB-Crys (W9F/W60F) lacking tryptophans, we have determined, through the fluorescence emission of substrate γD-Ctd tryptophans, that the γD-Ctd in the γD-Ctd_αB complexes is partially unfolded. The results are consistent with models in which the tryptophans normally buried in the Greek key, interact with the chaperone in the complex.

Chaperone Interactions of the Small Heat Shock Protein Human αb-Crystallin With Its Physiological Substrate γd-Crystallin and Its Isolated Domains

α-crystallin, a small heat shock protein chaperone, is one of the ubiquitous crystallins in the vertebrate lens, along with the βγ-crystallins. It is a polydisperse complex of ∼800 kD consisting of two subunits (∼20 kD) αA- and αB-crystallin (αA- and αB-Crys). Its chaperone activity involves suppressing aggregation by binding aggregation-prone species. Aggregates isolated from mature-onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of βγ-crystallins. The γ-crystallins are structural, monomeric proteins that consist of four Greek-key motifs organized into two domains. Human γD-crystallin (HγD-Crys) is a stable and long-lived mammalian γ-crystallin localized in the lens nucleus. It can refold in vitro to its native state after unfolding in high concentrations of GdnHCl. However, at very low denaturant concentrations (< 1 M GdnHCl) aggregation of refolding HγD-Crys intermediates competes with productive refolding. We have previously determined that the conformation of the bound HγD-Crys substrate in γD-αB complexes resembles the partially folded intermediate populated during refolding/unfolding equilibrium experiments, which has its N-terminal domain unfolded and its C-terminal domain folded. We have utilized single domain constructs to further characterize the binding interactions of HαB-Crys to different regions of HγD-Crys. The HγD-Crys C-terminal domain construct (γD-Ctd) aggregates upon refolding, while the N-terminal domain construct (γD-Ntd) does not aggregate under similar conditions. However, when γD-Ctd and γD-Ntd are unfolded and refolded together, γD-Ctd recruits γD-Ntd into the aggregate. HαB-Crys can suppress the aggregation of the γD-Ctd and forms γD-Ctd-αB complexes. Using a no-Trp mutant of HαB-Crys (W9F/W60F), we have determined, through the fluorescence emission of γD-Ctd tryptophans, that the γD-Ctd in the γD-Ctd-αB complexes is partially folded. These results provide further insight into how α-crystallin interacts with aggregation-prone substrates in vivo.

Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration.

Little is known about the role of protein dynamics in directing protein unfolding along a specific pathway and about the role played by chemical denaturants in modulating the dynamics and the initiation of unfolding. In this study, deuterium-hydrogen exchange (HX) detected by electrospray ionization mass spectrometry (ESI-MS) was used to study the unfolding of the SH3 domain of the PI3 kinase. Unfolding on the principal unfolding pathway occurs in 2 steps, both in the absence and in the presence of 1.8 M guanidine hydrochloride (GdnHCl). In both cases, the first step leads to the formation of an intermediate, I(N), with 5 fewer protected amide hydrogen sites than in N. In the second step, I(N) loses the structure protecting the remaining 14 amide hydrogen sites from HX as it unfolds completely. ESI-MS analysis of fragments of the protein created by proteolytic digestion, after completion of the HX reaction, shows that I(N) has lost protection against HX in the same segments of native structure during unfolding in the absence and presence of 1.8 M GdnHCl. Hence, GdnHCl does not appear to play a direct active role in the initiation of unfolding. However, at higher GdnHCl concentrations, a second unfolding pathway is shown to compete effectively with the N <--> I(N) <--> U pathway. In this way, the denaturant modulates the energy landscape of unfolding.

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 ϕ 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.

Human αB‐Crystallin suppresses the aggregation in vitro of partially folded intermediates of human γC‐, γD‐, and γS‐Crystallins

α‐crystallin, a small heat shock protein chaperone, is one of the ubiquitous crystallins in the vertebrate lens, along with the ??‐crystallins. It is a polydisperse complex of ~ 800 kD consisting of two subunits (~20 kD) αA‐ and αB‐crystallin (αA‐ and αB‐crys). Its chaperone activity involves suppressing aggregation by binding aggregation‐prone species. Aggregates isolated from mature‐onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of ??‐crystallins. Human ?D‐ and ?C‐crystallin (H?D‐ and H?C‐crys) are stable, long‐lived ?‐crystallins in the lens nucleus, while ?S‐crystallin (H?S‐crys) is abundant in the lens cortex. All three ?‐crystallins can refold in vitro to their native state after unfolding in high concentrations of GdnHCl. Refolding unfolded H?C‐, H?D‐, or H?S‐crys to very low GdnHCl concentrations (&lt; 1 M) resulted in partially folded intermediates partitioning between productive refolding and aggregation pathways. H?D‐, H?C‐ or H?S‐Crys protein was allowed to refold and aggregate in the presence of HαB‐Crys at different monomer‐to‐monomer ratios. H?D‐ and H?C‐Crys aggregation was suppressed to similar levels, whereas H?S‐Crys aggregation was not suppressed as well under identical conditions in assays measuring solution turbidity at 350 nm. These results provide a model for how α‐crystallin interacts with aggregation‐prone substrates in vivo

Revealing a Concealed Intermediate that Forms after the Rate-limiting Step of Refolding of the SH3 Domain of PI3 Kinase

Kinetic and equilibrium studies of the folding and unfolding of the SH3 domain of the PI3 kinase, have been used to identify a folding intermediate that forms after the rate-limiting step on the folding pathway. Folding and unfolding, in urea as well as in guanidine hydrochloride (GdnHCl), were studied by monitoring changes in the intrinsic fluorescence or in the far-UV circular dichroism (CD) of the protein. The two probes yield non-coincident equilibrium transitions for unfolding in urea, indicating that an intermediate, I, exists in equilibrium with native (N) and unfolded (U) protein, during unfolding. Hence, the equilibrium unfolding data were analyzed according to a three-state N ↔ I ↔ U mechanism. An intermediate is observed also in kinetic unfolding studies, and its presence leads to the unfolding reaction in urea as well as in GdnHCl, occurring in two steps. The fast step is complete within the initial 11 ms of unfolding and manifests itself in a burst phase change in fluorescence. At high concentrations of GdnHCl, the entire change in fluorescence during unfolding occurs during the 11 ms burst phase. CD measurements indicate, however, that I retains N-like secondary structure. An analysis of the kinetic and thermodynamic data, according to a minimal three-state N ↔ I ↔ U mechanism, positions I after the rate-limiting transition state, TS1, of folding, on the reaction coordinate of folding in GdnHCl. Hence, I is not revealed when folding is commenced from U, regardless of the nature of the probe used to follow the folding reaction. Interrupted unfolding experiments, in which the protein is unfolded transiently in GdnHCl for various lengths of time before being refolded, showed that I refolds to N much faster than does U, confirms the analysis of the direct folding and unfolding experiments, that I is formed after the rate-limiting step of refolding in GdnHCl.

HαB-Crystallin Suppresses The Aggregation Upon Refolding Of Its Physiological Substrates HγD-, HγC- And HγS-Crystallin

The passive chaperone α-crystallin, a small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses, along with the βγ-crystallins. It is composed of two subunits (∼ 20 kDa) αA- and αB-crystallin (αA- and αB-crys), which form an hetero-oligomeric, polydisperse complex of ∼ 800 kDa in the lens. Aggregates isolated from mature-onset cataracts, the major cause of sight loss worldwide, contain damaged and misfolded forms of βγ-crystallins, as well as α-crystallins. We have studied the chaperone function of Human αB-crystallin interacting with its physiological Human γ-crystallin substrates. Human γD-crystallin (HγD-crys) and γC-crystallin (HγC-crys) are stable and long-lived mammalian γ-crystallins localized in the lens nucleus. Human γS-crystallin (HγS-crys) is abundant in the lens outer cortex. All three γ-crystallins can refold in vitro to their native state after unfolding in high concentrations of GdnHCl. However, at very low denaturant concentrations (< 1 M GdnHCl) aggregation of refolding HγC- and HγD-Crys intermediates competes with productive refolding. Diluting unfolded HγC-, HγD-, or HγS-crys to low GdnHCl concentrations (100 μg/ml, 37°C) resulted in the protein population partitioning between productive refolding and aggregation pathways. HγD-, HγC- or HγS-Crys protein was allowed to refold and aggregate in the presence of HαB-Crys homo-oligomers at different monomer-to-monomer ratios of γ-Crys to αB-Crys. HγD- and HγC-Crys aggregation was suppressed to similar levels, whereas HγS-Crys aggregation was not suppressed as strongly in assays measuring solution turbidity at 350 nm. SEC chromatograms of the products of suppression reactions showed the presence of a high molecular weight complex containing the chaperone-substrate complex in ratios of 1γC:5αB and 1γD:5αB chains. This complex was still present 4 days after the suppression reaction was initiated. These results provide a model for how α-crystallin interacts with aggregation-prone substrates in vivo.

Steady state and time resolved effects of guanidine hydrochloride on the structure of Humicola lanuginosa lipase revealed by fluorescence spectroscopy.

Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild-type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady-state, stopped-flow, and time-resolved laser-induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped-flow fluorescence measurements.

Self-interaction of native and denatured lysozyme in the presence of osmolytes, l-arginine and guanidine hydrochloride

Abstract Osmolyte molecules such as betaine and trehalose are protein stabilizers while l -arginine (Arg) and guanidine hydrochloride (GdnHCl) are the most widely used aggregation suppressor in protein refolding. We have herein studied the effects of the osmolyte molecules and l -arginine together with GdnHCl (0–6 mol/L) on the intermolecular interaction of native and denatured lysozyme by self-interaction chromatography. The self-interaction is characterized in terms of the osmotic second virial coefficient (B) of the protein, the increase of which represents the decrease of intermolecular attraction of the protein. It is found that the effect of Arg on the self-interaction of lysozyme is similar with GdnHCl, but its competence is much weaker than the denaturant. At higher GdnHCl concentrations (>0.5 mol/L), Arg can be used to suppress the self-association of lysozyme. In contrast to Arg, B increases with increasing betaine or trehalose concentration at the GdnHCl concentration range studied. The results indicate the cooperativity of each osmolyte with GdnHCl, and the different mechanisms of their effects from Arg on the B values. The work confirms that the osmolytes are not only protein stabilizers, but also protein aggregation suppressors for both native and denatured protein molecules.

Folding of Escherichia coli DsbC: Characterization of a Monomeric Folding Intermediate

The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.

Conformation of Amyloid Fibrils of β2-Microglobulin Probed by Tryptophan Mutagenesis

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.

Intermediates in the inactivation and unfolding of dimeric arginine kinase induced by GdnHCl.

Equilibrium studies of guanidine hydrochloride (GdnHCl)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4 M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0 M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate I1; (2) sudden unwinding of I1 to another dimeric intermediate, I2; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4 M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5 M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5 M) to reconstitute and regain its catalytic activity completely.

Iron-Sulfur Cluster Biosynthesis

Important for the understanding of the functional properties of the iron-sulfur scaffold IscU is knowledge of the structure and dynamics of this protein class. Structural characterization of Thermotoga maritima IscU by CD (Mansy, S. S., Wu, G., Surerus, K. K., and Cowan, J. A. (2002) J. Biol. Chem. 277, 21397-21404) and high resolution NMR (Bertini, I., Cowan, J. A., Del Bianco, C., Luchinat, C., and Mansy, S. S. (2003) J. Mol. Biol. 331, 907-924) yielded data indicating a high degree of secondary structure. However, the latter also revealed IscU to exist in a dynamic equilibrium between two or more distinct conformations, possibly existing in a molten globule state. Herein, we further characterize the molten globule characteristics of T. maritima IscU by near-ultraviolet circular dichroism, 1-anilino-8-naphthalenesulfonic acid binding, free energy of unfolding, hydrodynamic radius measurements, and limited tryptic digestion. The data suggest unusual dynamic behavior that is not fully consistent with typical protein states such as fully folded, fully unfolded, or molten globule. For instance, the existence of a stable tertiary fold is supported by near-UV CD spectra and hydrodynamic radius measurements, whereas other data are less clearly interpretable and may be viewed as consistent with either a molten globule or fully folded state. However, all of the data are consistent with our previous hypothesis of a protein sampling multiple discrete tertiary conformations in which these structural transitions occur on a "slow" time scale. To describe such proteins, we introduce the term multiple discrete conformers.