A pH-dependent molten globule transition is required for activity of the steroidogenic acute regulatory protein, StAR

The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal alpha-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM.     

Membrane-protein interaction and the molten globule state: Interaction of α-lactalbumin with membranes

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovineα-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluoresence studies indicated rapid and tight binding of apo-α-lactalbumin (apo-α-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-α-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-α-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-α-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.     

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.     

Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.     

StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import

Cholesterol is the starting point for biosynthesis of steroids, oxysterols and bile acids, and is also an essential component of cellular membranes. The mechanisms directing the intracellular trafficking of this insoluble molecule have received attention through the discovery of the steroidogenic acute regulatory protein (StAR) and related proteins containing StAR-related lipid transfer domains. Much of our understanding of the physiology of StAR derives from studies of congenital lipoid adrenal hyperplasia, which is caused by StAR mutations. Multiple lines of evidence show that StAR moves cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. The precise mechanism by which StAR's action on the outer mitochondrial membrane stimulates the flow of cholesterol to the inner membrane remains unclear. When StAR interacts with protonated phospholipid head groups on the outer mitochondrial membrane, it undergoes a conformational change (molten globule transition) that opens and closes StAR's cholesterol-binding pocket; this conformational change is required for cholesterol binding, which is required for StAR activity. The action of StAR probably requires interaction with the peripheral benzodiazepine receptor.     

Membrane protein insertion regulated by bringing electrostatic and hydrophobic interactions into play. A case study with the translocation domain of diphtheria toxin

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.     

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of DeltaCp of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Retinol-binding protein is in the molten globule state at low pH.

Using far- and near-UV circular dichroism, viscosity, tryptophan fluorescence, NMR spectra, binding of a hydrophobic probe, and microcalorimetry, we have shown that the apo form of human retinol-binding protein (RBP) at neutral pH is in a rigid state with properties similar to those of holo-RBP. On the contrary, at acidic pH apo-RBP is in the molten globule state which has been earlier revealed for a number of proteins under mild denaturing conditions. We have also shown that, at equilibrium, the pH-induced retinol release from holo-RBP parallels denaturation of the apoprotein. These findings are consistent with our hypothesis that the transformation of RBP into the molten globule state is involved in the mechanism whereby retinol is delivered to target cells. In particular, a local acidic pH near the membrane surface of target cells might cause the transition of RBP to the molten globule state as well as the release of retinol.     

Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter

Cholesterol is a vital component of cellular membranes, and is the substrate for biosynthesis of steroids, oxysterols and bile acids. The mechanisms directing the intracellular trafficking of this nearly insoluble molecule have received increased attention through the discovery of the steroidogenic acute regulatory protein (StAR) and similar proteins containing StAR-related lipid transfer (START) domains. StAR can transfer cholesterol between synthetic liposomes in vitro, an activity which appears to correspond to the trans-cytoplasmic transport of cholesterol to mitochondria. However, trans-cytoplasmic cholesterol transport in vivo appears to involve the recently-described protein StarD4, which is expressed in most cells. Steroidogenic cells must also move large amounts of cholesterol from the outer mitochondrial membrane to the first steroidogenic enzyme, which lies on the matrix side of the inner membrane; this action requires StAR. Congenital lipoid adrenal hyperplasia, a rare and severe disorder of human steroidogenesis, results from mutations in StAR, providing a StAR knockout of nature that has provided key insights into its activity. Cell biology experiments show that StAR moves large amounts of cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. Biophysical data show that only the carboxyl-terminal alpha-helix of StAR interacts with the outer membrane. Spectroscopic data and molecular dynamics simulations show that StAR's interactions with protonated phospholipid head groups on the outer mitochondrial membrane induce a conformational change (molten globule transition) needed for StAR's activity. StAR appears to act in concert with the peripheral benzodiazepine receptor, but the precise itinerary of a cholesterol molecule entering the mitochondrion remains unclear.     

Exploring tryptophan dynamics in acid-induced molten globule state of bovine α-lactalbumin: a wavelength-selective fluorescence approach

The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning to be understood. Bovine α-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity (acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.     

Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

The interaction of adriamycin with small unilamellar vesicle liposomes. A fluorescence study

The interaction of the antineoplastic agent adriamycin with sonicated liposomes composed of phosphatidylcholine alone and with small amounts (1-6%) of cardiolipin has been studied by fluorescence techniques. Equilibrium binding data show that the presence of cardiolipin increases the amount of drug bound to liposomes when the bilayer is below its phase transition temperature and when the ionic strength is relatively low (0.01 M). At higher ionic strength (0.15 M) and above the Tm (i.e. conditions which are closer to the physiological state) the binding of the drug to the two liposome types is nearly the same. Thus the differences in the interactions of adriamycin with cardiolipin-containing membranes, as opposed to those composed of phosphatidylcholine alone, are not due simply to increased binding but rather to an altered membrane structure when this lipid is present. Quenching of adriamycin fluorescence by iodide shows that bound drug is partially, but not completely, buried in the liposomal membrane. Both in the presence and absence of cardiolipin the bulk of the adriamycin is more accessible to the quencher below the Tm than above it; that is, a solid membrane tends to exclude the drug from deep penetration. Above the Tm, the presence of cardiolipin alters the nature of liposome-adriamycin interaction. Here the fluorescence quenching data suggest that the presence of small amounts of cardiolipin (3%) in a phosphatidylcholine matrix creates two types of binding environments for drug, one relatively exposed and the other more deeply buried in the membrane. The temperature dependence of the adriamycin fluorescence and the liposome light scattering reveal that cardiolipin alters the thermal properties of the bilayer as well as its interaction with adriamycin. At low ionic strength lateral phase separations may occur with both pure phosphatidylcholine and when 3% cardiolipin is present; under these conditions the bound adriamycin exists in two kinds of environment. It is notable that only adriamycin fluorescence reveals this phenomenon; thebulk property of liposome light scattering reports only on the overall membrane phase change. These data suggest that under certain conditions the drug binding sites in the membranes are decoupled from the bulk of the lipid bilayer.     

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.     

Hydrophobic core variant ubiquitin forms a molten globule conformation at acidic pH

The conformational properties of hydrophobic core variant ubiquitin (Val26 to Ala mutation) in an acidic solution were studied. The intrinsic tryptophan fluorescence emission spectrum, far-UV and near-UV circular dichroic spectra, the fluorescence emission spectrum of 8-anilinonaphthalene-1-sulfonic acid in the presence of V26A ubiquitin, and urea-induced unfolding measurements indicate this variant ubiquitin to be in the partially folded molten globule conformation in solution at pH 2. The folding kinetics from molten globule to the native state was nearly identical to those from the unfolded state to the native state. This observation suggests that the equilibrium molten globule state of hydrophobic core variant ubiquitin is an on-pathway folding intermediate.     

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

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

Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.