Heat denaturation of human orosomucoid in water/methanol mixtures

Heat denaturation of orosomucoid in solutions of methanol concentrations ranging from 0 to 70% (v/v) has been studied by using circular dichroism, intrinsic protein fluorescence and thermal difference absorption spectroscopy. Regardless of its high saccharide content (40%), the highly cooperative denaturation transition of orosomucoid is fully reversible in neutral water solution. A two-state model has been successfully applied; the numerical analysis results in thermodynamical parameter values that are in close agreement with previously reported experimental data from calorimetric measurements. However, in solutions containing even minute concentrations of methanol (5%) the heat denaturation is irreversible. After cooling of the denatured protein the refolded molecules exhibit a higher α-helical content than the native one. Possibilities of methanol interaction with native and denatured protein molecule are discussed.     

Intermediates in the refolding of reduced ribonuclease A.

The intermediates with one, two, three or four disulphide bonds which accumulate during unfolding of native ribonuclease and refolding of the reduced protein have been trapped by rapid alkylation with iodoacetate and separated by ionexchange chromatography. They have been characterized to varying extents by their enzymic activity, electrophoretic mobility through polyacrylamide gels, disulphide bonds between cysteine residues, the environments of the six tyrosine residues as indicated by ultraviolet absorption and fluorescence spectra, interaction with antibodies directed against either the trapped unfolded reduced protein or the native folded protein, and for the disruption by urea of any stable conformation producing a change in molecular shape.Correctly refolded ribonuclease was indistinguishable from the original native protein, but virtually all the intermediates with up to four disulphide bonds formed directly from the reduced protein were enzymically inactive and unfolded by these criteria. Unfolding of native ribonuclease was an all-or-none transition to the fully reduced protein, with no accumulation of disulphide intermediates. The intermediates in refolding are separated from the fully folded state by the highest energy barrier in the folding transition; they may be considered rapidly interconvertible, relatively unstable microstates of the unfolded protein. The measured elements of the final conformation are not acquired during formation of the first three disulphide bonds, but appear simultaneously with formation of the fourth native disulphide bond.These observations with ribonuclease are qualitatively similar to those made previously in greater detail with pancreatic trypsin inhibitor and suggest a possible general pattern for the kinetic process of protein unfolding and refolding.     

Stable,metastable, and supercritical phases in solutions of globular proteins between upper and lower denaturation temperatures

The temperature trends of the standard thermodynamic functions of the native and denatured protein in solution are considered within the concept of excess mixing functions. It is assumed that some protein molecules adopt an intermediate state between native and denatured forms within the temperature range between cold and thermal denaturation and form metastable microphases as a result of a specific interaction with water. A phase diagram in the temperature–standard entropy coordinate plane representing an isobar family is proposed. Two limiting isobars are characterized by an entropy jump, which reflects the first-order phase transition between the native and denatured states. The isobars in the intermediate temperature range are represented as van der Waals curves, which reflect the equilibrium between the main phase of the molecules in native state and microphases. The difference between the phases disappears at critical points. It is assumed that the supercritical range is a macroscopically homogeneous single phase zone of reduced stability, which is represented by a dynamic system of monomers and oligomers of the native protein, monomers and clusters of the protein with partially unfolded structure. The phase diagram is collated with the elliptic phase diagram in the temperature–osmotic pressure plane.     

Quantitative analysis of the composition of the native and scrambled ribonuclease A

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2-1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.     

Ester-linked hen egg white lysozyme shows a compact fold in a molecular dynamics simulation - possible causes and sensitivity of experimentally observable quantities to structural changes maintaining this compact fold

Prediction and understanding of the folding and stability of the 3D structure of proteins is still a challenge. The different atomic interactions, such as non polar contacts and hydrogen bonding, are known but their exact relative weights and roles when contributing to protein folding and stability are not identified. Initiated by a previous molecular dynamics simulation of fully ester-linked hen egg white lysozyme (HEWL), which showed a more compact fold of the ester-linked molecule compared to the native one, three variants of this protein are analyzed in the present study. These are 129-residue native HEWL, partly ester-linked HEWL, in which only 34 peptide linkages that are not involved in the helical or β-strand parts of native HEWL were replaced by ester linkages, and fully (126 residues) ester-linked HEWL. Native and partly ester-linked HEWL showed comparable behaviour, whereas fully ester-linked HEWL could not maintain the native secondary structure of HEWL in the simulation and adopted a more compact fold. The conformational changes were analyzed by comparing simulation averaged values of quantities that can be measured by NMR, such as (1)H-(15)N backbone order parameters, residual dipolar couplings, proton-proton NOE distances and (3)J-couplings with the corresponding values derived from experimental NMR data for native HEWL. The information content of the latter appeared to be insufficient to detect the local conformational rearrangements upon esterification of the loop regions of the protein. For fully ester-linked HEWL, a significantly reduced agreement was observed. Upon esterification, the backbone-side chain and side chain-side chain hydrogen-bonding pattern of HEWL changes to maintain its compactness and thus the structural stability of the ester-linked lysozymes.     

Molecular dynamics simulation of thionated hen egg white lysozyme

Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited.     

Comparison of the folding processes of distantly related proteins. Importance of hydrophobic content in folding

The N-terminal domain of HypF from Escherichia coli (HypF-N) is a 91 residue protein module sharing the same folding topology and a significant sequence identity with two extensively studied human proteins, muscle and common-type acylphosphatases (mAcP and ctAcP). With the aim of learning fundamental aspects of protein folding from the close comparison of so similar proteins, the folding process of HypF-N has been studied using stopped-flow fluorescence. While mAcP and ctAcP fold in a two-state fashion, HypF-N was found to collapse into a partially folded intermediate before reaching the fully folded conformation. Formation of a burst-phase intermediate is indicated by the roll over in the Chevron plot at low urea concentrations and by the large jump of intrinsic and 8-anilino-1-naphtalenesulphonic acid-derived fluorescence immediately after removal of denaturant. Furthermore, HypF-N was found to fold rapidly with a rate constant that is approximately two and three orders of magnitudes faster than ctAcP and mAcP, respectively. Differences between the bacterial protein and the two human counterparts were also found as to the involvement of proline isomerism in their respective folding processes. The results clearly indicate that features that are often thought to be relevant in protein folding are not highly conserved in the evolution of the acylphosphatase superfamily. The large difference in folding rate between mAcP and HypF-N cannot be entirely accounted for by the difference in relative contact order or related topological metrics. The analysis shows that the higher folding rate of HypF-N is in part due to the relatively high hydrophobic content of this protein. This conclusion, which is also supported by the highly significant correlation found between folding rate and hydrophobic content within a group of proteins displaying the topology of HypF-N and AcPs, suggests that the average hydrophobicity of a protein sequence is an important determinant of its folding rate.     

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol.

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42 degrees C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Th1) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-delta Tm 16 degrees C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a beta-strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.     

Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum.

Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium glutamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative allosteric effector isoleucine, the K0.5 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. glutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology. Therefore, the C. glutamicum enzyme must be considered a small variant of threonine dehydratase that is typically controlled by isoleucine and valine but has an altered structure reflecting a topological difference in the portion of the protein most likely to be important for allosteric regulation.     

Native mass spectrometry: a bridge between interactomics and structural biology

Native mass spectrometry is an emerging technology that allows the topological investigation of intact protein complexes with high sensitivity and a theoretically unrestricted mass range. This unique tool provides complementary information to established technologies in structural biology, and also provides a link to high-throughput interactomics studies, which do not generate information on exact protein complex-composition, structure or dynamics. Here I review the current state of native mass spectrometry technology and discuss several important biological applications. I also describe current experimental challenges in native mass spectrometry, encouraging readers to contribute to solutions.     

Topological determinants of protein unfolding rates

For proteins that fold by two-state kinetics, the folding and unfolding processes are believed to be closely related to their native structures. In particular, folding and unfolding rates are influenced by the native structures of proteins. Thus, we focus on finding important topological quantities from a protein structure that determine its unfolding rate. After constructing graphs from protein native structures, we investigate the relationships between unfolding rates and various topological quantities of the graphs. First, we find that the correlation between the unfolding rate and the contact order is not as prominent as in the case of the folding rate and the contact order. Next, we investigate the correlation between the unfolding rate and the clustering coefficient of the graph of a protein native structure, and observe no correlation between them. Finally, we find that a newly introduced quantity, the impact of edge removal per residue, has a good overall correlation with protein unfolding rates. The impact of edge removal is defined as the ratio of the change of the average path length to the edge removal probability. From these facts, we conclude that the protein unfolding process is closely related to the protein native structure.     

Topological information content and chemical reactions

The difference of the topological information content of two reacting molecules and that of their reaction products is calculated for several topological types of chemical reactions, illustrating the influence of the structure of the reagents and of the reaction product. It is shown that the change in the topological information content in a chemical reaction can be positive as well as negative, depending on the way the reagents approach each other and thus on the reaction product formed. A quantitative measure of structural specificity is introduced.     

Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins.

The burial of native disulfide bonds, formed within stable structure in the regeneration of multi-disulfide-containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol-disulfide exchange reactions. Nevertheless, several proteins retain solvent-exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40-95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild-type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40-95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi-disulfide-containing protein.     

Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link.

The authors in a previous report (Klausner, R. D., Kempf, C., Weinstein, J. N., Blumenthal, R., and van Renswoude, J. (1983) Biochem. J. 212, 801-810) have argued that native folding of ovalbumin occurs during translation, but not in a renaturation system of the denatured form. To re-examine the possibility, we searched for the conditions of correct oxidative refolding of denatured disulfide-reduced ovalbumin. Data of trypsin resistance, CD-spectrum, and selective reactivity of cysteine sulfhydryls revealed that the fully denatured protein can refold into the native conformation under disulfide-reduced conditions. The interconversion between the native and denatured forms was fully reversible with a free energy change for unfolding of 6.6 kcal/mol at 25 degrees C. Subsequent reoxidation under a variety of redox conditions generated only one disulfide bond in the reduced refolded protein with six cysteine sulfhydryls. Furthermore, the regenerated disulfide was found by peptide analyses to correspond to the native disulfide pairing, Cys73-Cys120. We, therefore, concluded that co-translational folding, if any, is not requisite for the correct oxidative folding of ovalbumin.     

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.     

The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation

The refolding and reoxidation of fully reduced and denatured chymotrypsinogen A have been studied in the presence of low concentrations of guanidine HCl or urea. Renaturation yields of 60 to 70% were observed when the reoxidation was facilitated by mixtures of reduced and oxidized glutathione. Refolding occurred within a narrow range of denaturant concentration (1.0 to 1.3 M guanidine HCl and 2 M urea) in which the native protein was shown to be stable, and the reduced protein was shown to regain the correct disulfide pairing. Renatured chymotrypsinogen is indistinguishable from the native zymogen in chromatographic behavior, potential chymotryptic activity, sedimentation coefficient, and spectral properties. The kinetics of renaturation were determined. Some of the protein species obtained at various times of renaturation were characterized as incorrectly oxidized molecules which could be renatured by thiol-catalyzed interchange of disulfide bonds.     

NMR structural characterization and computational predictions of the major intermediate in oxidative folding of leech carboxypeptidase inhibitor

The III-A intermediate constitutes the major rate-determining step in the oxidative folding of leech carboxypeptidase inhibitor (LCI). In this work, III-A has been directly purified from the folding reaction and structurally characterized by NMR spectroscopy. This species, containing three native disulfides, displays a highly native-like structure; however, it lacks some secondary structure elements, making it more flexible than native LCI. III-A represents a structurally determined example of a disulfide-insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We also show that theoretical approaches based on topological constraints predict with good accuracy the presence of this folding intermediate. Overall, the derived results suggest that, as it occurs with non-disulfide bonded proteins, native-like interactions between segments of secondary structure rather than the crosslinking of disulfide bonds direct the folding of LCI.     

Lipid-dependent Generation of Dual Topology for a Membrane Protein

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. We posit that such duality is determined by both the protein sequence and the membrane lipid composition wherein a spatial or temporal change in the latter can result in a post-assembly change in protein structure and function. To investigate whether co-existence of multiple topological conformers is dependent on the membrane lipid composition, we determined the topological organization of lactose permease in an Escherichia coli model cell system in which phosphatidylethanolamine membrane content can be systematically varied. At intermediate levels of phosphatidylethanolamine a mixture of native and topologically mis-oriented conformers co-existed. There was no threshold level of phosphatidylethanolamine determining a sharp transition from one conformer to the other. Co-existing conformers were not in rapid equilibrium at a static lipid composition indicating that duality of topology is established during an early folding step. Depletion of intermediate levels of phosphatidylethanolamine after final protein assembly resulted in complete mis-orientation of the native conformer. Combined with previous results, such topological dynamics are reversible in both directions. We propose a thermodynamically based model for how lipid-protein interactions can result in a mixed topological organization and how changes in lipid composition can result in changes in the ratio of topologically distinct conformers of proteins. These observations demonstrate a potential lipid-dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within the same membrane or between different membranes in more complex eukaryotic cells.     

A new topological method to measure protein structure similarity

A method for the quantitative evaluation of structural similarity between protein pairs is developed that makes use of a Delaunay-based topological mapping. The result of the mapping is a three-dimensional array which is representative of the global structural topology and whose elements can be used to construe an integral scoring scheme. This scoring scheme was tested for its dependence on the protein length difference in a pairwise comparison, its ability to provide a reasonable means for structural similarity comparison within a family of structural neighbors of similar length, and its sensitivity to the differences in protein conformation. It is shown that such a topological evaluation of similarity is capable of providing insight into these points of interest. Protein structure comparison using the method is computationally efficient and the topological scores, although providing different information about protein similarity, correlate well with the distance root-mean-square deviation values calculated by rigid-body structural alignment.