Elucidation of stable intermediates in urea-induced unfolding pathway of human carbonic anhydrase IX

Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0–4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located β-sheets. The reduction of 60–65% tertiary contacts in 7.0–8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.     

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra

The urea-induced unfolding of staphylococcal nuclease A has been studied by circular dichroism both at equilibrium and by the kinetics of unfolding and refolding (pH 7.0 and 4.5 degrees C), as a function of Ca2+ and thymidine 3',5'-diphosphate (pdTp) concentration. The results are as follows. (1) The unfolding transition is shifted to higher concentrations of urea by Ca2+ and pdTp, and the presence of both ligands further stabilizes the protein. (2) In the first stage of kinetic refolding, the peptide ellipticity changes rapidly within the dead time of stopped-flow measurement (15 ms), indicating accumulation of a transient intermediate. This intermediate is remarkably less stable than those of other globular proteins previously studied. (3) Dependence of the folding and unfolding rate constants on urea concentration indicates that the critical activated state of folding ("transition state") has considerable structural organization. The transition state does not, however, have the capacity to bind Ca2+ and pdTp, as indicated by the effects of these ligands on the unfolding rate constant. (4) There are at least four different phases in the refolding kinetics in native conditions below 1 M urea. In the absence of pdTp, there are two phases in unfolding, while in the presence of pdTp the unfolding kinetics show a single phase. Some characteristics of the transient intermediate and of the transition state for folding are discussed.     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.     

Hydrophobic core of molten-globule state of bovine carbonic anhydrase B

The interaction which stabilizes the intermediate state of the protein folding and/or unfolding is important for understanding the structure formation mechanism of proteins. The partitioning of a hydrophobic fluorescence probe, pyrene, into the core of a ‘molten globule’ structure of bovine carbonic anhydrase B was measured, revealing a partition coefficient of about 10⁴. The result leads to the conclusion that the compact structure of the molten-globule state is formed by the hydrophobic interaction, as detergent micelles are formed by the same interaction.     

Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.     

Carbonic Anhydrase B Interactions with Water and Urea

High-resolution NMR spectroscopy has been used to study native carbonic anhydrase B unfolding with urea at pH 5.75 and T = 298 K. The rigidity parameter reflecting the effectiveness of spin diffusion (SD) displays a sigma-like dependence on urea concentration, which is characteristic of denaturing processes. The ratio between the integral intensities of urea and protein signals measured in SD spectra and normal 1D spectra are the same. This suggests the absence of a predominant interaction between urea and protein molecules. The concentration of large protein-solvent complexes rapidly increases at urea concentrations of 4.2–6.2 M, which is apparently related to the transition of the protein into the molten globule state. If the urea concentration is increased to 6.6 M, these complexes dissociate, and the polypeptide chain of carbonic anhydrase B becomes completely unfolded.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 497–503.Original Russian Text Copyright © 2005 by Prokhorov, Kutyshenko, Khristoforov.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

Equilibrium and kinetics of the unfolding and refolding of Escherichia coli Malate Synthase G monitored by circular dichroism and fluorescence spectroscopy

The equilibrium and kinetics studies of an 82 kDa large monomeric Escherichia coli protein Malate Synthase G (MSG) was investigated by far and near-UV CD, intrinsic tryptophan fluorescence and extrinsic fluorescence spectroscopy. We find that despite of its large size, folding is reversible, in vitro. Equilibrium unfolding process of MSG exhibited three-state transition thus, indicating the presence of at least a stable equilibrium intermediate. Thermodynamic parameters suggest this intermediate resembles the unfolded state. However, the equilibrium intermediate exhibits pronounced secondary structure as measured by far-UV CD, partial tertiary structure as delineated by near-UV CD, compactness (m value) and exposed hydrophobic surface area as assessed by ANS binding, typically depicting a molten globule state. The stopped-flow kinetic data provide clear evidence for the presence of a burst phase during the refolding pathway due to the formation of an early Intermediate, within the dead time of the instrument. Refolding from 4 M to various lower concentrations until 0.4 M of GdnHCl follow biphasic kinetics at lower concentrations of GdnHCl (<0.8 M), whereas monophasic kinetics at concentrations above 1.5 M. Also, rollover in the refolding and unfolding limbs of chevron plot verifies the presence of a fast kinetic intermediate at lower concentration of GdnHCl. Based upon the above observations we hereby propose the folding pathway of a large multi-domain protein Malate Synthase G.     

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.     

Evidence for a molten globule state as a general intermediate in protein folding

The folding of globular proteins occurs through intermediate states whose characterisation provides information about the mechanism of folding. A major class of intermediate states is the compact 'molten globule', whose characteristics have been studied intensively in those conditions in which it is stable (at acid pH, high temperatures and intermediate concentrations of strong denaturants). In studies involving bovine carbonic anhydrase, human alpha-lact-albumin, bovine beta-lactoglobulin, yeast phosphoglycerate kinase, beta-lactamase from Staphylococcus aureus and recombinant human interleukin 1 beta, we have demonstrated that a transient intermediate which accumulates during refolding is compact and has the properties of the 'molten globule' state. We show that it is formed within 0.1-0.2 s. These proteins belong to different structural types (beta, alpha + beta and alpha/beta), with and without disulphide bridges and they include proteins with quite different times of complete folding (from seconds to decades of minutes). We propose that the formation of the transient molten globule state occurs early on the pathway of folding of all globular proteins.     

Fluctuating state of the protein globule

It has been shown that bovine and human alpha-lactalbumins and carbonic anhydrase B can be transformed under different influence into a peculiar state possessing physical characteristics intermediate between those for the native and unfolded states. In this state a protein molecule is compact and has the secondary structure similar to that of the native molecule, but does not melt cooperatively at heating, has an anomalously fast H-D exchange and a more or less symmetrical average environment of aromatic and other side groups. A model of this "intermediate" state of protein molecule is proposed, according to which the intermediate state differs from the native one mainly by the substantial increase of protein structure fluctuations and the sharp decrease of van der Waals and other specific interactions. It has been shown that the transition from the native state to this intermediate state is the phase transition of the first order. The role of the intermediate state in protein folding is discussed.     

Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates.The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.     

Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0–6 M at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.     

Trehalose favors a cutinase compact intermediate off-folding pathway

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Comparison of NMR structural and dynamics features of the urea and guanidine-denatured states of GED

Denatured states of proteins, the starting points as well as the intermediates of folding in vivo, play important roles in biological function. In this context, we describe here urea unfolding and characterization of the denatured state of GTPase effector domain (GED) of dynamin created by 9.7 M urea. These are compared with similar data for guanidine induced denaturation reported earlier. The unfolding characteristics in the two cases, as measured by the optical probes, are significantly different, urea unfolding proceeding via an intermediate. The structural and motional characteristics, determined by NMR, of the two denatured states are also strikingly different. The urea-denatured state shows a combination of α- and β-preferences in contrast to the entirely β-preferences in the guanidine-denatured state. Higher ¹⁵N transverse relaxation rates suggest higher folding propensities in the urea-denatured state. The implications of these to GED folding are discussed.     

Beta-sheet proteins with nearly identical structures have different folding intermediates

The folding mechanisms of two proteins in the family of intracellular lipid binding proteins, ileal lipid binding protein (ILBP) and intestinal fatty acid binding protein (IFABP), were examined. The structures of these all-beta-proteins are very similar, with 123 of the 127 amino acids of ILBP having backbone and C(beta) conformations nearly identical to those of 123 of the 131 residues of IFABP. Despite this structural similarity, the sequences of these proteins have diverged, with 23% sequence identity and an additional 16% sequence similarity. The folding process was completely reversible, and no significant concentrations of intermediates were observed by circular dichroism or fluorescence at equilibrium for either protein. ILBP was less stable than IFABP with a midpoint of 2. 9 M urea compared to 4.0 M urea for IFABP. Stopped-flow kinetic studies showed that both the folding and unfolding of these proteins were not monophasic, suggesting that either multiple paths or intermediate states were present during these processes. Proline isomerization is unlikely to be the cause of the multiphasic kinetics. ILBP had an intermediate state with molten globule-like spectral properties, whereas IFABP had an intermediate state with little if any secondary structure during folding and unfolding. Double-jump experiments showed that these intermediates appear to be on the folding path for each protein. The folding mechanisms of these proteins were markedly different, suggesting that the different sequences of these two proteins dictate different paths through the folding landscape to the same final structure.