Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

How, when and why proteins collapse: the relation to folding

Unfolded proteins under strongly denaturing conditions are highly expanded. However, when the conditions are more close to native, an unfolded protein may collapse to a compact globular structure distinct from the folded state. This transition is akin to the coil-globule transition of homopolymers. Single-molecule FRET experiments have been particularly conducive in revealing the collapsed state under conditions of coexistence with the folded state. The collapse can be even more readily observed in natively unfolded proteins. Time-resolved studies, using FRET and small-angle scattering, have shown that the collapse transition is a very fast event, probably occurring on the submicrosecond time scale. The forces driving collapse are likely to involve both hydrophobic and backbone interactions. The loss of configurational entropy during collapse makes the unfolded state less stable compared to the folded state, thus facilitating folding.     

Denatured proteins and early folding intermediates simulated in a reduced conformational space

Conformations of globular proteins in the denatured state were studied using a high-resolution lattice model of proteins and Monte Carlo dynamics. The model assumes a united-atom and high-coordination lattice representation of the polypeptide conformational space. The force field of the model mimics the short-range protein-like conformational stiffness, hydrophobic interactions of the side chains and the main-chain hydrogen bonds. Two types of approximations for the short-range interactions were compared: simple statistical potentials and knowledge-based protein-specific potentials derived from the sequence-structure compatibility of short fragments of protein chains. Model proteins in the denatured state are relatively compact, although the majority of the sampled conformations are globally different from the native fold. At the same time short protein fragments are mostly native-like. Thus, the denatured state of the model proteins has several features of the molten globule state observed experimentally. Statistical potentials induce native-like conformational propensities in the denatured state, especially for the fragments located in the core of folded proteins. Knowledge-based protein-specific potentials increase only slightly the level of similarity to the native conformations, in spite of their qualitatively higher specificity in the native structures. For a few cases, where fairly accurate experimental data exist, the simulation results are in semiquantitative agreement with the physical picture revealed by the experiments. This shows that the model studied in this work could be used efficiently in computational studies of protein dynamics in the denatured state, and consequently for studies of protein folding pathways, i.e. not only for the modeling of folded structures, as it was shown in previous studies. The results of the present studies also provide a new insight into the explanation of the Levinthal's paradox.     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Hierarchic organization of domains in globular proteins

An automatic procedure is developed for the identification of domains in globular proteins from X-ray elucidated co-ordinates. Using this tool, domains are shown to be iteratively decomposable into subdomains, leading to a hierarchic molecular architecture.There is no convenient geometry that will fully characterize the atom by atom interdigitation at an interface between domains, and the strategy adopted here was devised to reduce this unwieldy three-dimensional problem to a closely approximating companion analysis in a plane. These analytically derived domain choices can be used subsequently to construct computer-generated, space-filling, color-coded views of the domains; and when this is done, the derived domains are seen to be completely resolved.The number of domains in a protein is a mathematically well-behaved function of the chain length, lending support to the supposition that the domains are an implicit structural consequence of the folding process. A spectrum of domains ranging in size from whole protein monomers to the individual units of secondary structure is apparent in each of the 22 proteins analyzed here.The hierarchic organization of structural domains is evidence in favor of an underlying protein folding process that proceeds by hierarchic condensation. In this highly constrained model, every pathway leading to the native state can be described by a tree of local folding interactions.     

Local interactions and the optimization of protein folding

The role of local interactions in protein folding has recently been the subject of some controversy. Here we investigate an extension of Zwanzig's simple and general model of folding in which local and nonlocal interactions are represented by functions of single and multiple conformational degrees of freedom, respectively. The kinetics and thermodynamics of folding are studied for a series of energy functions in which the energy of the native structure is fixed, but the relative contributions of local and nonlocal interactions to this energy are varied over a broad range. For funnel shaped energy landscapes, we find that 1) the rate of folding increases, but the stability of the folded state decreases, as the contribution of local interactions to the energy of the native structure increases, and 2) the amount of native structure in the unfolded state and the transition state vary considerably with the local interaction strength. Simple exponential kinetics and a well-defined free energy barrier separating folded and unfolded states are observed when nonlocal interactions make an appreciable contribution to the energy of the native structure; in such cases a transition state theory type approximation yields reasonably accurate estimates of the folding rate. Bumps in the folding funnel near the native state, which could result from desolvation effects, side chain freezing, or the breaking of nonnative contacts, significantly alter the dependence of the folding rate on the local interaction strength: the rate of folding decreases when the local interaction strength is increased beyond a certain point. A survey of the distribution of strong contacts in the protein structure database suggests that evolutionary optimization has involved both kinetics and thermodynamics: strong contacts are enriched at both very short and very long sequence separations. Proteins 29:282–291, 1997. © 1997 Wiley-Liss, Inc.     

Ligand binding to cytochrome c and other related haem proteins and peptides. Part I. Equilibrium studies

Investigation of ligand binding to native cytochrome c, carboxymethyl-Met 80-cytochrome c, myoglobin and haemhexapeptide revealed that the binding of exogenous ligands is modulated by the following factors:

• 1.Hydrophobicity of the haem environment.
• 2.Haem accessibility to exogenous ligands, termed the haem crevice ‘open-closed’ parameter.
• 3.Steric interactions between the protein and the bound ligand.

     

The Origin of Nonmonotonic Complex Behavior and the Effects of Nonnative Interactions on the Diffusive Properties of Protein Folding

We present a method for calculating the configurational-dependent diffusion coefficient of a globular protein as a function of the global folding process. Using a coarse-grained structure-based model, we determined the diffusion coefficient, in reaction coordinate space, as a function of the fraction of native contacts formed Q for the cold shock protein (TmCSP). We find nonmonotonic behavior for the diffusion coefficient, with high values for the folded and unfolded ensembles and a lower range of values in the transition state ensemble. We also characterized the folding landscape associated with an energetically frustrated variant of the model. We find that a low-level of frustration can actually stabilize the native ensemble and increase the associated diffusion coefficient. These findings can be understood from a mechanistic standpoint, in that the transition state ensemble has a more homogeneous structural content when frustration is present. Additionally, these findings are consistent with earlier calculations based on lattice models of protein folding and more recent single-molecule fluorescence measurements.     

Non-carbohydrate binding partners/domains of animal lectins

• 1.1. Protein-carbohydrate interactions are involved in a large number of biologically important recognition processes.
• 2.2. Among the participating classes of proteins lectins are defined as carbohydrate-binding proteins other than an antibody or an enzyme.
• 3.3. In addition to the essential carbohydrate-binding domain other functionally and/or structurally important sites, defined by sequence comparison or by experimental demonstration of protein-protein interactions, can be present within the lectin molecule and may be relevant for its physiological significance.
• 4.4. Sequence motifs of lectins for protein-protein interactions include amino acid structures designed for cell adhesion, growth regulatory biosignalling, intracellular routing and enzymatic activity.
• 5.5. Elucidation of the complete functional role(s) of a lectin requires accurate delineation of its carbohydrate and, if present, of its protein ligands.
• 6.6. Presence of more than one carbohydrate-binding domain in a single lectin, potential ligand properties of the glycopart of a lectin, regulatory interplay between different sites and possible interaction of complementarily shaped peptide sequences to the sugar-recognizing site should all be assessed in the quest to comprehensively explain the physiological role(s) of a lectin.

     

Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain

The N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) state and a relatively compact unfolded (U(exch)) state under nondenaturing conditions. Selectively labeled samples of the domain have been analyzed by NOESY NMR experiments to probe residual hydrophobic clustering in the U(exch) state. The labeling strategy included selective protonation of aromatic rings or delta-methyl groups on Ile and Leu residues in a highly deuterated background. Combined with long mixing times, the methods permitted observation of significant numbers of long-range interactions between hydrophobic side chains, providing evidence for multiple conformers involving non-native hydrophobic clusters around the Trp 36 indole. Comparison of these data with previously reported HN-HN NOEs yields structural insight into the diversity of structures within the U(exch) ensemble in the drkN SH3 domain. Many of the HN-HN NOEs are consistent with models containing compact residual nativelike secondary structure and greater exposure of the Trp 36 indole to solvent, similar to kinetic intermediates formed in the hierarchic condensation model of folding. However, the methyl and aromatic NOE data better fit conformations with non-native burial of the Trp indole surrounded by hydrophobic groups and more loosely formed beta-structure; these structural characteristics are more consistent with those of kinetic intermediates formed during the hydrophobic collapse mechanism of folding. This suite of NOE data provides a more complete picture of the structures that span the U(exch) state ensemble, from conformers with non-native structure but long-range contacts to those that are highly nativelike. Together, the results are also consistent with the folding funnel view involving multiple folding pathways for this molecule.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H

Elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. Here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the Thermus thermophilus ribonuclease H, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an α-helix and two β-strands (51 folded residues). Using multidimensional NMR, we solved the structure of this intermediate mimic to an atomic resolution (backbone rmsd, 0.51 Å). It has a native-like backbone topology and shows some local deviations from the native structure, revealing that the structure of the folded region of an early folding intermediate can be as well defined as the native structure. The topological parameters calculated from the structures of the intermediate mimic and the native state predict that the intermediate should fold on a millisecond time scale or less and form much faster than the native state. Other factors that may lead to the slow folding of the native state and the accumulation of the intermediate before the rate-limiting transition state are also discussed.     

Regulation by progesterone and pregnenolone of dimeric aldehyde dehydrogenase from rat testis cytoplasm

• 1.1. Aldehyde dehydrogenase from rat testis cytosol has been purified to electrophoretic homogeneity. With an isoelectric point of 9.5, the enzyme appears a dimer with a subunit molecular weight of 52,500.
• 2.2. The influence of pregnenolone and progesterone on the kinetic behaviour has been investigated using valeraldehyde as substrate.
• 3.3. The kinetic data were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of valeraldehyde concentrations.
• 4.4. According to the model, the dimeric enzyme is in equilibrium between two confonnational states R and T. The R state displays higher affinity for valeraldehyde, but lower catalytic power. In the absence of substrates and effectors the [T]/[R] ratio is near to 1.
• 5.5. Pregnenolone and progesterone activate the enzyme by stabilizing the more active state T and by increasing the catalytic power of the R state. The increase of activity is counteracted by the inhibition exerted by both steroids on the T state.

     

The interaction of human serum albumin in the native and fully reduced states with apolipoprotein E,serum amiloid protein and very low density lipoproteins from human plasma

• 1.1. Complex formation in a solution of apolipoprotein E (apoE) isolated from human plasma very low density lipoproteins (VLDL) and human serum albumin (HSA) in both native and fully reduced states was studied. The existence of a kinetically unstable complex of apoE and native albumin was shown. The complex became more stable with the reduction of the S—S links in the albumin molecules capable of forming aggregates under these conditions.
• 2.2. The interaction between native HSA as opposed to a fully reduced one and isolated VLDL particles was more pronounced, probably, due to the existence of amphipathic alpha-helical regions.
• 3.3. Dissociation of the serum amyloid protein (SAP) oligomeric form in solution and the interaction of the protein with fully reduced HSA owing to the provision with the additional hydrophobic surface was shown. ApoE displaced SAP from the complex with fully reduced albumin.
• 4.4. It is suggested that the ability of the apolipoprotein to interact with albumin is determined by internal stability of the molecular structure of the latter and the complexes detected in vitro may be a new transport form of apolipoproteins in lipid-free form in serum. It is assumed that competitive interactions in the HSA-SAP-apoE system may be involved in the development of secondary amyloidosis.

     

Purification and characterization of the d-lactate dehydrogenase from Leuconostoc lactis

• 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
• 2.2.The enzyme is a dimer of subunits of M_r = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
• 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.

     

Purification and some particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.

     

Structured pathway across the transition state for peptide folding revealed by molecular dynamics simulations

Small globular proteins and peptides commonly exhibit two-state folding kinetics in which the rate limiting step of folding is the surmounting of a single free energy barrier at the transition state (TS) separating the folded and the unfolded states. An intriguing question is whether the polypeptide chain reaches, and leaves, the TS by completely random fluctuations, or whether there is a directed, stepwise process. Here, the folding TS of a 15-residue β-hairpin peptide, Peptide 1, is characterized using independent 2.5 μs-long unbiased atomistic molecular dynamics (MD) simulations (a total of 15 μs). The trajectories were started from fully unfolded structures. Multiple (spontaneous) folding events to the NMR-derived conformation are observed, allowing both structural and dynamical characterization of the folding TS. A common loop-like topology is observed in all the TS structures with native end-to-end and turn contacts, while the central segments of the strands are not in contact. Non-native sidechain contacts are present in the TS between the only tryptophan (W11) and the turn region (P7-G9). Prior to the TS the turn is found to be already locked by the W11 sidechain, while the ends are apart. Once the ends have also come into contact, the TS is reached. Finally, along the reactive folding paths the cooperative loss of the W11 non-native contacts and the formation of the central inter-strand native contacts lead to the peptide rapidly proceeding from the TS to the native state. The present results indicate a directed stepwise process to folding the peptide.     

Growth rates and body condition factors of Alligator mississippiensis in coastal Louisiana wetlands: A comparison of wild and farm-released juveniles

• 1.1. Growth rates and body condition factors for native wild and captive-raised juvenile alligators (Alligator mississippiensis) that had been released to the wild were studied using tag-recapture methods for 274 alligators over a 4-year period. Alligators were grouped by sex, size class, source (farm-released vs native wild) and as to whether they had overwintered or not.
• 2.2. In most groups, the farm-released alligators grew significantly better than wild alligators matched for sex and size; in the remaining groups the post-release alligators grew as well as their counterparts, though not better.
• 3.3. Overwintering tended to slow growth rates in both groups, but farm-released alligators still demonstrated superior growth over native wild alligators even after overwintering.
• 4.4. Males tended to grow faster than females, though this trend was not always significantly greater. In no matched group did females grow faster than males.
• 5.5. Growth rates diminished with increasing size in native wild alligators (smaller alligators grew faster), but growth rates of farm-released alligators remained accelerated even at the larger size classes.
• 6.6. Growth curves were constructed using known recapture data with three growth models (von Bertlanffy, Gompertz and logistic); the calculated maximum attainable length and growth parameters were significantly larger (P < 0.01) for farm-released alligators than wild using all three models.
• 7.7. Body condition factors were not different in captive-raised post-released alligators than native wild alligators.

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.