Radius of gyration is indicator of compactness of protein structure

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Statistical analysis of radii of gyration for 3769 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha + beta) demonstrates that each class of proteins has its own class-specific radius of gyration, which determines compactness of protein structures: alpha-proteins have the largest radius of gyration. This indicates that they are less tightly packed than beta- and alpha + beta-proteins. Finally, alpha/beta-proteins are the most tightly packed proteins with the least radius of gyration. It should be underlined that radius of gyration normalized on the radius of gyration of ball with the same volume, is independent of the length in comparison with such parameters as compactness and number of contacts per residue.     

Conformational analysis of polypeptides and proteins for the study of protein folding, molecular recognition, and molecular design

Conformational energy calculations provide an understanding as to how interatomic interactions lead to the three-dimensional structures of polypeptides and proteins, and how these molecules interact with other molecules. Illustrative results of such calculations pertain to model systems (-helices and -sheets, and interactions between them), to various open-chain and cyclic peptides, to fibrous proteins, to globular proteins, and to enzyme-substrate complexes. In most cases, the validity of the computations is established by experimental tests of the predicted structures.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985. This paper first appeared in the Israel Journal of Chemistry, Vol. 27, 1986.     

Accurate prediction of protein structural classes using functional domains and predicted secondary structure sequences

Protein structural class prediction is one of the challenging problems in bioinformatics. Previous methods directly based on the similarity of amino acid (AA) sequences have been shown to be insufficient for low-similarity protein data-sets. To improve the prediction accuracy for such low-similarity proteins, different methods have been recently proposed that explore the novel feature sets based on predicted secondary structure propensities. In this paper, we focus on protein structural class prediction using combinations of the novel features including secondary structure propensities as well as functional domain (FD) features extracted from the InterPro signature database. Our comprehensive experimental results based on several benchmark data-sets have shown that the integration of new FD features substantially improves the accuracy of structural class prediction for low-similarity proteins as they capture meaningful relationships among AA residues that are far away in protein sequence. The proposed prediction method has also been tested to predict structural classes for partially disordered proteins with the reasonable prediction accuracy, which is a more difficult problem comparing to structural class prediction for commonly used benchmark data-sets and has never been done before to the best of our knowledge. In addition, to avoid overfitting with a large number of features, feature selection is applied to select discriminating features that contribute to achieve high prediction accuracy. The selected features have been shown to achieve stable prediction performance across different benchmark data-sets.     

Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications

A homodimer of pyruvate dehydrogenase kinase (PDHK) is an integral part of pyruvate dehydrogenase complex (PDC) to which it is anchored primarily through the inner lipoyl-bearing domains (L2) of transacetylase component. The catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the "symmetric" and "asymmetric" modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively. Whereas the structure of the symmetric PDHK/L2 complex was reported, the structural organization and functional role of the asymmetric complex remain obscure. Here, we report the crystal structure of the asymmetric PDHK3/L2 complex that reveals several functionally important features absent from the previous structures. First, the PDHK3 subunits have distinct conformations: one subunit exhibits "open" and the other "closed" configuration of the putative substrate-binding cleft. Second, access to the closed cleft is additionally restricted by local unwinding of the adjacent alpha-helix. Modeling indicates that the target peptide might gain access to the PDHK active center through the open but not through the closed cleft. Third, the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive "pre-insertion" binding mode. Altogether our data suggest that the asymmetric complex represents a physiological state in which binding of a single L2-domain activates one of the PDHK protomers while inactivating another. Thus, the L2-domains likely act not only as the structural anchors but also modulate the catalytic cycle of PDHK.     

Massive sequence perturbation of the Raf ras binding domain reveals relationships between sequence conservation, secondary structure propensity, hydrophobic core organization and stability

The contributions of specific residues to the delicate balance between function, stability and folding rates could be determined, in part by [corrected] comparing the sequences of structures having identical folds, but insignificant sequence homology. Recently, we have devised an experimental strategy to thoroughly explore residue substitutions consistent with a specific class of structure. Using this approach, the amino acids tolerated at virtually all residues of the c-Raf/Raf1 ras binding domain (Raf RBD), an exemplar of the common beta-grasp ubiquitin-like topology, were obtained and used to define the sequence determinants of this fold. Herein, we present analyses suggesting that more subtle sequence selection pressure, including propensity for secondary structure, the hydrophobic core organization and charge distribution are imposed on the Raf RBD sequence. Secondly, using the Gibbs free energies (DeltaG(F-U)) obtained for 51 mutants of Raf RBD, we demonstrate a strong correlation between amino acid conservation and the destabilization induced by truncating mutants. In addition, four mutants are shown to significantly stabilize Raf RBD native structure. Two of these mutations, including the well-studied R89L, are known to severely compromise binding affinity for ras. Another stabilized mutant consisted of a deletion of amino acid residues E104-K106. This deletion naturally occurs in the homologues a-Raf and b-Raf and could indicate functional divergence. Finally, the combination of mutations affecting five of 78 residues of Raf RBD results in stabilization of the structure by approximately 12 kJ mol(-1) (DeltaG(F-U) is -22 and -34 kJ mol(-1) for wt and mutant, respectively). The sequence perturbation approach combined with sequence/structure analysis of the ubiquitin-like fold provide a basis for the identification of sequence-specific requirements for function, stability and folding rate of the Raf RBD and structural analogues, highlighting the utility of conservation profiles as predictive tools of structural organization.     

Vanadate as an inhibitor of plant and mammalian peroxidases

Vanadate ions are shown to inhibit horseradish, squash, and rat intestinal peroxidases by following the reaction spectrophotometrically in a wide range of vanadate concentrations. I₅₀ in phosphate buffer were 43, 9.4, and 535 μM, respectively. No inhibitory effect was found on cow milk lactoperoxidase and beef liver catalase. Gel filtration of peroxidases in the presence of vanadate, as carried out by radioactive⁴⁸V for horseradish peroxidases (either in aerobic or anoxic conditions) and neutron activation analysis (NAA) for squash peroxidase, demonstrated a binding of vanadium to these enzymes in stoichiometric amounts. Electron paramagnetic resonance spectra of the eluted peaks for the former peroxidase indicated that vanadium is in the +5 oxidation state, but an equilibrium between V (V) and V (IV) in the assay conditions cannot be discarded. Although the inhibitory mechanism remains obscure, some hypotheses are considered. The potential implications that the inhibitory effect of vanadium might have on plant and animal metabolism are also discussed.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production.     

A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein

Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I(BP), and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of I(BP) and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on I(BP) and I1 for replacements in alpha3, beta4 and alpha8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I(BP) reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)beta(3) core in I(BP). The retention of I(BP) and the smaller and consistent destabilization of both I(BP) and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-the-barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I(BP) or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM barrel and other members of the (beta/alpha)(n) class of proteins.     

Novel structure of the conserved gram-negative lipopolysaccharide transport protein A and mutagenesis analysis

Lipopolysaccharide (LPS) transport protein A (LptA) is an essential periplasmic localized transport protein that has been implicated together with MsbA, LptB, and the Imp/RlpB complex in LPS transport from the inner membrane to the outer membrane, thereby contributing to building the cell envelope in Gram-negative bacteria and maintaining its integrity. Here we present the first crystal structures of processed Escherichia coli LptA in two crystal forms, one with two molecules in the asymmetric unit and the other with eight. In both crystal forms, severe anisotropic diffraction was corrected, which facilitated model building and structural refinement. The eight-molecule form of LptA is induced when LPS or Ra-LPS (a rough chemotype of LPS) is included during crystallization. The unique LptA structure represents a novel fold, consisting of 16 consecutive antiparallel β-strands, folded to resemble a slightly twisted β-jellyroll. Each LptA molecule interacts with an adjacent LptA molecule in a head-to-tail fashion to resemble long fibers. Site-directed mutagenesis of conserved residues located within a cluster that delineate the N-terminal β-strands of LptA does not impair the function of the protein, although their overexpression appears more detrimental to LPS transport compared with wild-type LptA. Moreover, altered expression of both wild-type and mutated proteins interfered with normal LPS transport as witnessed by the production of an anomalous form of LPS. Structural analysis suggests that head-to-tail stacking of LptA molecules could be destabilized by the mutation, thereby potentially contributing to impair LPS transport.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

Toward protein structure in situ: comparison of two bifunctional rhodamine adducts of troponin C

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca(2+)-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.     

Investigation of the cumulative tissue doses of naphthoquinones in human serum using protein adducts as biomarker of exposure

Both 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are reactive metabolites of naphthalene that are thought to be responsible for the naphthalene-induced cytotoxicity and genotoxicity. The aim of this study was to investigate the cumulative tissue dose of 1,2-NPQ and 1,4-NPQ in human serum derived from blood donors in Taiwan via measurements of albumin adducts by a methodology, which employs trifluoroacetic acid anhydride and methanesulfonic acid to selectively cleave cysteinyl adducts on proteins. Both 1,2-NPQ and 1,4-NPQ adducts were detected in all male and female subjects (n = 22). The median levels of 1,2-NPQ adduct in human subjects were estimated to be 268 (range 139-857) and 203 (range 128-1352) (pmol/g) in male (n = 11) and female (n = 11) subjects, respectively. In contrast, the median levels of 1,4-NPQ adduct were estimated to be 45.0 (range 22.0-117) and 38.9 (range 21.5-172) (pmol/g) in male and female subjects, respectively. We noticed that levels of 1,2-NPQ adduct were significantly correlated with those of 1,4-NPQ adduct (correlation coefficient r = 0.643, p < 0.01). Results from in vitro experiments confirmed that the production of naphthoquinones-derived adducts on serum albumin increased with increased concentration of naphthoquinones (0-100 μM). Linear relationships were observed over the range of concentration. Time-course experiments suggested that both 1,2-NPQ and 1,4-NPQ-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter. The reaction rate constant analyses indicated that the second-order rate constants, representing in vitro reactions between naphthoquinones and cysteine residues of serum albumin, were estimated to be 0.0044/0.0002 L(g protein)⁻¹ h⁻¹, respectively. Overall, the cumulative tissue doses of 1,4-NPQ (217-316 nM h) in male and female subjects were ∼3-fold greater than those of 1,2-NPQ (76-98 nM h) in the study population. The initial concentrations of serum 1,2-NPQ and 1,4-NPQ in the study population were estimated to be between 145-188 and 807-1175 nM, respectively. We conclude that the relatively large amounts of naphthoquinones present in human serum may point to toxicological consequences.     

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death

F-Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N-terminal F-box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp-1/Cul1/F-Box) E3-ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C-termini using either leucine rich repeats (LRR) or tryptophan-aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F-Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3-ubiquitin ligase that targets the multifunctional regulator Y-box binding protein 1 (YB-1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB-1 for proteasomal degradation, FBX33 negatively interferes with YB-1 mediated functions. In contrast to typical FBPs, FBX33 has no C-terminal LRR or WD40 domains and associates with YB-1 via its N-terminus. The present study confirms the existence of a formerly hypothetical F-Box protein in living cells and describes one of its substrates.     

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.