Biomedical subjects

András Perczel

Publications and source records attributed to András Perczel.

At least 19 recordsLinked to original sources

PubMed2006

Prediction of folding preference of 10 kDa silk-like proteins using a Lego approach and ab initio calculations.

Because of their great flexibility and strength resistance, both spider silks and silkworm silks are of increasing scientific and commercial interest. Despite numerous spectroscopic and theoretical studies, several structural properties at the atomic level have yet to be identified. The present theoretical investigation focuses on these issues by studying three silk-like model peptides: (AG)(64), [(AG)(4)EG](16), and [(AG)(4)PEG](16), using a Lego-type approach to construct these polypeptides. On the basis of these examples it is shown that thermoneutral isodesmic reactions and ab initio calculations provide a capable method to investigate structural properties of repetitive polypeptides. The most probable overall fold schema of these molecules with respect to the type of embedded hairpin structures were determined at the ab initio level of theory (RHF/6-311++G(d,p)//RHF/3-21G). Further on, analysis is carried out on the possible hairpin and turn regions and on their effect on the global fold. In the case of the (AG)(64) model peptide, the optimal beta-sheet/turn ratio was also determined, which provided good support for experimental observations. In addition, lateral shearing of a hairpin "folding unit" was investigated at the quantum chemical level to explain the mechanical properties of spider silk. The unique mechanical characteristics of silk bio-compounds are now investigated at the atomic level.

Models, Molecular↗

PubMed2006

Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein.

A 10 kDa dynein light chain (DLC), previously identified as a tail light chain of myosin Va, may function as a cargo-binding and/or regulatory subunit of both myosin and dynein. Here, we identify and characterize the binding site of DLC on myosin Va. Fragments of the human myosin Va tail and the DLC2 isoform were expressed, and their complex formation was analyzed by pull-down assays, gel filtration, and spectroscopic methods. DLC2 was found to bind as a homodimer to a approximately 15 residue segment (Ile1280-Ile1294) localized between the medial and distal coiled-coil domains of the tail. The binding region contains the three residues coded by the alternatively spliced exon B (Asp1284-Lys1286). Removal of exon B eliminates DLC2 binding. Co-localization experiments in a transfected mammalian cell line confirm our finding that exon B is essential for DLC2 binding. Using circular dichroism, we demonstrate that binding of DLC2 to a approximately 85 residue disordered domain (Pro1235-Arg1320) induces some helical structure and stabilizes both flanking coiled-coil domains (melting temperature increases by approximately 7 degrees C). This result shows that DLC2 promotes the assembly of the coiled-coil domains of myosin Va. Nuclear magnetic resonance spectroscopy and docking simulations show that a 15 residue peptide (Ile1280-Ile1294) binds to the surface grooves on DLC2 similarly to other known binding partners of DLCs. When our data are taken together, they suggest that exon B and its associated DLC2 have a significant effect on the structure of parts of the coiled-coil tail domains and such a way could influence the regulation and cargo-binding function of myosin Va.

Alternative Splicing↗

PubMed2006

A self-stabilized model of the chymotrypsin catalytic pocket. The energy profile of the overall catalytic cycle.

A model of the catalytic triad of chymotrypsin is built assuring the arrangement and properties as they are within the complete enzyme. The model contains 18 amino acid residues of chymotrypsin and its substrate. A total of 135 atoms (including 70 heavy atoms) were subjected to full ab initio geometry optimizations through 127 individual steps along the reaction coordinate of the complete catalytic mechanism. It was shown that the described model of the catalytic apparatus forms a self-stabilized molecule ensemble without the rest of the enzyme and substrate. According to the calculations, the formations of the first and second tetrahedral intermediates in the model have 20.3 and 15.7 kcal/mol activation energy barriers, respectively. Removing elements of the catalytic apparatus such as the (1) catalytic aspartate or (2) the anion hole, as well as (3) inserting a water molecule "early" in the catalytic process, or (4) introducing conformational rigidity of the substrate, results in an increase of the above energy barrier of the first catalytic step in the model by 6.4, 13.7, 3.7, and 4.1 kcal/mol, respectively. Based on the calculated process one can conclude that the catalytic reaction in this model is much more similar to the reaction in the enzyme than to the reference reaction. To our knowledge, this is the first model system that mimics the complete catalytic mechanism.

Binding Sites↗

PubMed2006

Determining suitable lego-structures to estimate stability of larger peptide nanostructures using computational methods.

Nanofibers, nanofilms and nanotubes constructed of one to four strands of oligo-alpha- and oligo-beta-peptides were obtained by using carefully selected building units. Lego-type approaches based on thermoneutral isodesmic reactions can be used to reconstruct the total energies of both linear and tubular periodic nanostructures with acceptable accuracy. Total energies of several different nanostructures were accurately determined with errors typically falling in the subchemical range. Thus, attention will be focused on the description of suitable isodesmic reactions that have enabled the determination of the total energy of polypeptides and therefore offer a very fast, efficient and accurate method to obtain energetic information on large and even very large nanosystems.

Algorithms↗

PubMed2006

Toward a rational design of beta-peptide structures.

Intrinsic conformational characteristics of beta-peptides built up from simple achiral and chiral beta-amino acid residues (i.e., HCO-beta-Ala-NH2, HCO-beta-Abu-NH2) were studied using quantum chemical calculations and 1H-NMR spectroscopy. A conformer-based systematic and uniform nomenclature was introduced to differentiate conformers. Geometry optimizations were performed on all homoconformers of both HCO-(beta-Ala)(k)-NH2 and HCO-(beta-Abu)(k)-NH2 (1 < or = k < or = 6) model systems at the RHF/3-21G and RHF/6-311++G(d, p) levels of theory. To test for accuracy and precision, additional computations were carried out at several levels of theory [e.g., RHF/6-31G(d), and B3LYP/6-311++G(d, p)]. To display the folding preference, the relative stability of selected conformers as function of the length of the polypeptide chain was determined. Ab initio population distribution of hexapeptides and the conformational ensemble of synthetic models composed of beta-Ala and beta-Abu studied using 1H-NMR in different solvents were compared at a range of temperatures. Helical preference induced by various steric effects of nonpolar side chains was tested using higher level ab initio methods for well-known model systems such as: HCO-(beta-HVal-beta-HAla-beta-HLeu)2-NH2, HCO-(ACHC)6-NH2, HCO-(trans-ACPC)6-NH2, and HCO-(cis-ACPC)6-NH2. The relative stabilities determined by theoretical methods agreed well with most experimental data, supporting the theory that the local conformational preference influenced by steric effects is a key determining factor of the global fold both in solution and in the gas phase.

Magnetic Resonance Spectroscopy↗

PubMed2006

Local binding with globally distributed changes in a small protease inhibitor upon enzyme binding.

Complexation of the small serine protease inhibitor Schistocerca gregaria chymotrypsin inhibitor (SGCI), a member of the pacifastin inhibitor family, with bovine chymotrypsin was followed by NMR spectroscopy. (1)H-(15)N correlation (HSQC) spectra of the inhibitor with increasing amounts of the enzyme reveal tight and specific binding in agreement with biochemical data. Unexpectedly, and unparalleled among canonical serine protease inhibitors, not only residues in the protease-binding loop of the inhibitor, but also some segments of it located spatially far from the substrate-binding cleft of the enzyme were affected by complexation. However, besides changes, some of the dynamical features of the free inhibitor are retained in the complex. Comparison of the free and complexed inhibitor structures revealed that most, but not all, of the observed chemical shift changes can be attributed to minor structural transitions. We suggest that the classical 'scaffold + binding loop' model of canonical inhibitors might not be fully valid for the inhibitor family studied. In our view, this feature allows for the emergence of both taxon-specific and nontaxon-specific inhibitors in this group of small proteins.

Amino Acid Sequence↗

PubMed2005

Extended intermolecular interactions in a serine protease-canonical inhibitor complex account for strong and highly specific inhibition.

We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use.

Amino Acid Sequence↗

PubMed2005

Peptide models XLV: conformational properties of N-formyl-L-methioninamide and its relevance to methionine in proteins.

The conformational space of the most biologically significant backbone folds of a suitable methionine peptide model was explored by density functional computational method. Using a medium [6-31G(d)] and a larger basis set [6-311++G(2d,2p)], the systematic exploration of low-energy backbone structures restricted for the "L-region" in the Ramachandran map of N-formyl-L-methioninamide results in conformers corresponding to the building units of an extended backbone structure (betaL), an inverse gamma-turn (gammaL), or a right-handed helical structure (alphaL). However, no poly-proline II type (epsilonL) fold was found, indicating that this conformer has no intrinsic stability, and highlighting the effect of molecular environment in stabilizing this backbone structure. This is in agreement with the abundance of the epsilonL-type backbone conformation of methionine found in proteins. Stability properties (DeltaE) and distinct backbone-side-chain interactions support the idea that specific intramolecular contacts are operative in the selection of the lowest energy conformers. Apart from the number of different folds, all stable conformers are within a 10 kcal x mol(-1) energy range, indicating the highly flexible behavior of methionine. This conformational feature can be important in supporting catalytic processes, facilitating protein folding and dimerization via metal ion binding. In both of the biological examples discussed (HIV-1 reverse transcriptase and PcoC copper-resistant protein), the conformational properties of Met residues were found to be of key importance. Spatial proximity to other types of residues or the same type of residue seems to be crucial for the structural integrity of a protein, whether Met is buried or exposed.

Amino Acids↗

PubMed2005

Structure and stability of beta-pleated sheets.

Beside alpha-helices, beta-sheets are the most common secondary structure elements of proteins. In this article, the question of structure and stability of parallel and antiparallel sheets of various lengths is addressed. All data obtained are compared to a selected set of protein structures. In antiparallel beta-sheets, one of the two possible H-bonded structures (containing 14 atoms in the H-bonded pseudoring) is energetically more favored and also more abundant in proteins than the other one (with 10 atoms involved in the pseudoring). Parallel beta-sheets and their subunits are energetically less stable and indeed found to occur more rarely in proteins. Antiparallel hairpins are disfavored compared to beta-sheets formed by sequentially separated strands. Agreement between theory and experimental data indicates that characterization of structural building blocks at an appropriately accurate level of theory is a useful tool to get insight into fundamentals of protein structure.

Alanine↗

PubMed2005

Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI: chemical shift analysis of his to gain 3D structure and protonation state information.

NMR--chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N'-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[(1)H(alpha)]/phi, CSI[(13)C(alpha)]/phi, and CSI[(13)C(alpha)]/psi. Protonation of the imidazole ring induces the upfield shift of CSI[(13)C(alpha)] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental (13)C(alpha), (13)C(beta), and (1)H(alpha) chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse gamma-turns) and loops beyond the assignment of chemical shifts to alpha-helices and beta-pleated sheets. Moreover, the location of the His residue can be further specified in a beta-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last beta-strand within a beta-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., (1)H(alpha) and (13)C(alpha)) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced.

Amino Acids↗

PubMed2005

Solution state conformation and degradation of cyclopeptides containing an NGR motif.

In contrast to the RGD-peptides, head to tail cyclization of LNGRV and LNGRv caused only a marginal change in their integrin receptor affinity as shown by the limited effect and selectivity on the adhesion of endothelial cells to ECM components. Structure determination of the two cyclopeptides by NMR and MD, semiempirical and ab initio methods revealed that both are very flexible and take on multiple stable conformers in solution. This structural diversity, along with the presence of the Asn-Gly peptide bond, enhances succinimide ring formation leading to the hydrolysis of Asn. It has been demonstrated that c(LNGRV) suffers deamidation with time both in solution and during storage. As the isoaspartyl-peptide may co-elute with the asparginyl-peptide in the course of HPLC analysis, MS measurement is necessary to check the purity of peptides containing the NGR sequence. Our stability investigations raise the question whether the NGR motif or its hydrolysis product is effective in in vivo experiments.

Amino Acid Motifs↗

PubMed2004

The structure of the complex of calmodulin with KAR-2: a novel mode of binding explains the unique pharmacology of the drug.

3'-(beta-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca(2+)-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation ((1)H-(15)N heteronuclear single quantum coherence) spectra of (15)N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.

Animals↗

PubMed2004

Binding crevice for TT-232 in a homology model of type 1 somatostatin receptor.

Somatostatin receptor type 1 was modelled based on the atomic structure of bovine rhodopsin. Possible ways of binding interaction between somatostatin receptor type 1 and TT-232, a cycloheptapeptide analogue of somatostatin with broad therapeutic potential, were analysed by molecular docking. The twelve TT-232 conformations, obtained by NMR measurements in H(2)O-D(2)O mixture, were similar, disclosing a consensus backbone conformation. Several residues interacting with TT-232, such as Val133, Asp137 (helix 3), Arg197 (helix 4), Phe287, Gln291, Asn294 (helix 6), Ser305, and Tyr313 (helix 7), were found. In accordance, in vitro binding experiments indicated high-affinity binding of TT-232 to (125)I labelled somatostatin sites in brain membranes. The single binding crevice obtained by docking may allow the design and discovery of new peptidomimetics of TT-232 in the future.

Amino Acid Sequence↗

PubMed2004

Vicinal disulfide bridge conformers by experimental methods and by ab initio and DFT molecular computations.

A systematic comparison is made between experimental and computational data gained on vicinal disulfide bridges in proteins and peptides. Structural and stability data of ab initio and density functional theory (DFT) calculations on the model compound 4,5-ditiaheptano-7-lactam and the model peptide HCO-ox-[Cys-Cys]-NH2 at RHF/3-21G*, B3LYP/6-31+G(d), and B3LYP/6-311++G(d,p) levels of theory are presented. The data on Xxx-Cys-Cys-Yyy type amino acid sequence units retrieved from PDB SELECT, along with data on sequence units that have vicinal disulfide bridge, taken from the Brookhaven Protein Data Bank, are conformationally characterized. Amino acid backbone conformations, cis-trans isomerism of the amide bond between the two cysteine residues, and ring puckering are studied. Ring puckers are characterized by their relation to the conformers of the parent 4,5-ditiaheptano-7-lactam. Computational precision and accuracy are proved by frequency calculation and solvent model optimization on selected conformers. It is found that the ox-[Cys-Cys] unit is able to accept types I, II, VIa, VIb, and VIII beta-turn structures.

Amino Acids↗

PubMed2004

Same fold with different mobility: backbone dynamics of small protease inhibitors from the desert locust, Schistocerca gregaria.

SGCI (Schistocerca gregaria chymotrypsin inhibitor) and SGTI (Sch. gregaria trypsin inhibitor) are small, 35-residue serine protease inhibitors with intriguing taxon specificity: SGTI is specific for arthropod proteases while SGCI is an excellent inhibitor on both mammalian and arthropodal enzymes. Here we report the cloning, expression, and (15)N backbone dynamics investigations of these peptides. Successful expression could be achieved by a "dimeric" construct similar to the natural precursor of the inhibitors. An engineered methionine residue between the two modules served as a unique cyanogen bromide cleavage site to cleave the precursor and physically separate SGCI and SGTI. The overall correlation time of the precursor (5.29 ns) as well as the resulted SGCI (3.14 ns) and SGTI (2.96 ns) are as expected for proteins of this size. General order parameters (S(2)) for the inhibitors are lower than those characteristic of well-folded proteins. Values in the binding loop region are even lower. Interestingly, the distribution of residues for which a chemical exchange (R(ex)) term should be considered is strikingly different in SGCI and SGTI. Together with H-D exchange studies, this indicates that the internal dynamics of the two closely related molecules differ. We suggest that the dynamic properties of these inhibitors is one of the factors that determine their specificity.

Amino Acid Sequence↗

PubMed2004

On the flexibility of beta-peptides.

The full conformational space was explored for an achiral and two chiral beta-peptide models: namely For-beta-Ala-NH2, For-beta-Abu-NH2, and For-beta-Aib-NH2. Stability and conformational properties of all three model systems were computed at different levels of theory: RHF/3-21G, B3LYP/6-311++G(d,p)//RHF/3-21G, B3LYP/6-311++G(d,p), MP2//B3LYP/6-311++G(d,p), CCSD//B3LYP/6-311++G(d,p), and CCSD(T)//B3LYP/6-311++G(d,p). In addition, ab initio E = E(phi, micro, psi) potential energy hypersurfaces of all three models were determined, and their topologies were analyzed to determine the inherent flexibility properties of these beta-peptide models. Fewer points were found and assigned than expected on the basis of Multidimensional Conformational Analysis (MDCA). Furthermore, it has been demonstrated, that the four-dimensional surface, E = E(phi, mu, psi), can be reduced into a three-dimensional one: E = E[phi, f(phi), psi]. This reduction of dimensionality of freedom of motion suggests that beta-peptides are less flexible than one would have thought. This agrees with experimental data published on the conformational properties of peptides composed of beta-amino acid residues.

Models, Molecular↗

PubMed2004

A simple fold with variations: the pacifastin inhibitor family.

Members of the pacifastin family are small, approximately 35-residue serine protease inhibitors isolated from arthropod species. Several locust inhibitors exhibit intriguing taxon specificity while others do not. The structural basis of this phenomenon may lie in the different dynamical properties of the proteins originating from different stabilizing interactions. In this study, we identify new members of the family to confirm the universal role of these interactions in the family. Structural investigations show that both the disulfide pattern and the stabilizing interactions are unique among small all-beta proteins.

Binding Sites↗

PubMed2004

Multidimensional NMR identifies the conformational shift essential for catalytic competence in the 60-kDa Drosophila melanogaster dUTPase trimer.

The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible for the prevention of uracil incorporation into DNA, involves ordering of the flexible C terminus of the enzyme. This conformational shift is investigated by multidimensional NMR on the Drosophila enzyme. Flexible segments of the homotrimer give rise to sharp resonances in the (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, which are clearly distinguishable from the background resonances of the well folded protein globule. Binding of the product dUMP or the analogues dUDP and alpha,beta-imino-dUTP to the enzyme induces a conformational change reflected in the disappearance of eight sharp resonances. This phenomenon is interpreted as nucleotide binding-induced ordering of some residues upon the folded protein globule. Three-dimensional (15)N-edited (1)H-(15)N HSQC total correlation spectroscopy (TOCSY) and (1)H-(15)N HSQC nuclear Overhauser effect spectroscopy measurements allowed clear assignment of these eight specific resonance peaks. The residues identified correspond to the conserved C-terminal sequence motif, indicating that (i) this conformational shift is amenable to NMR studies in solution even in the large trimeric molecule and (ii) formation of the closed enzyme conformer in the case of the Drosophila enzyme does not require the complete triphosphate chain of the substrate. NMR titration of the enzyme with the nucleotide ligands as well as kinetic data indicated significant deviation from the model of independent active sites within the homotrimer. The results suggest allosterism in the eukaryotic dUTPase.

Allosteric Site↗