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For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound KRas. The identified low-populated higher energy state in GDPloaded K-Ras has a conformation reminiscent of a nucleotidebound/Mg2+-free state characterized by shortened β2/β3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutationspecific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Pyranuronic β-sugar amino acids (β-SAAs) are biocompatible and tuneable building blocks of foldamers and chimera-peptides. The scalable and economical total synthesis of two building blocks is described here. These C-4 epimers, Fmoc-GlcAPU(Me)-OH (7) and Fmoc-GalAPU(Me)-OH (8), which are suitable for solid phase peptide synthesis, were prepared via a common oxime intermediate 16. The new synthesis uses nine consecutive steps, starting from methyl α-d-glucopyranoside (6). The synthesis is fine-tuned, optimized, and ready for large-scale and cost-efficient production.

Non-covalent interactions between ions and aromatic rings play an important role in the stabilization of macromolecular complexes;of particular interest are peptides and proteins containing aromatic side chains (Phe, Trp, and Tyr) interacting with negatively (Asp and Glu) and positively (Arg and Lys) charged amino acid residues. The structures of the ion-aromatic-ring complexes are the result of an interaction between the large quadrupole moment of the ring and the charge of the ion. Four attractive interaction types are proposed to be distinguished based on the position of the ion with respect to the plane of the ring: perpendicular cation-pi (CP perpendicular to), co-planar cation-pi (CP parallel to), perpendicular anion-pi (AP(perpendicular to)), and co-planar anion-pi (AP(parallel to)). To understand more than the basic features of these four interaction types, a systematic, high-level quantum chemical study is performed, using the X- + C6H6, M+ + C6H6, X- + C6F6, and M+ +C6F6 model systems with X- = H-, F-, Cl-, HCOO-, CH3COO- and M- = H+, Li-, Na+, NH4+, CH3NH3+, whereby C6H6 and C6F6 represent an electron-rich and an electron-deficient pi system, respectively. Benchmark-quality interaction energies with small uncertainties, obtained via the so-called focal-point analysis (FPA) technique, are reported for the four interaction types. The computations reveal that the interactions lead to significant stabilization, and that the interaction energy order, given in kcal mol(-1) in parentheses, is CP perpendicular to (23-37)> AP(perpendicular to) (14-21)> CP parallel to (9-22)> AP(parallel to) (6-16). A natural bond orbital analysis performed leads to a deeper qualitative understanding of the four interaction types. To facilitate the future quantum chemical characterization of ion-aromatic-ring interactions in large biomolecules, the performance of three density functional theory methods, B3LYP, BH and HLYP, and M06-2X, is tested against the FPA benchmarks, with the result that the M06-2X functional performs best. (C) 2017 Wiley Periodicals, Inc.

Both far- and near-UV electronic circular dichroism (ECD) spectra have bands sensitive to thermal unfolding of Trp and Tyr residues containing proteins. Beside spectral changes at 222 nm reporting secondary structural variations (far-UV range), Lb bands (near-UV range) are applicable as 3D-fold sensors of protein's core structure. In this study we show that both Lb (Tyr) and Lb (Trp) ECD bands could be used as sensors of fold compactness. ECD is a relative method and thus requires NMR referencing and cross-validation, also provided here. The ensemble of 204 ECD spectra of Trp-cage miniproteins is analysed as a training set for "calibrating" Trp↔Tyr folded systems of known NMR structure. While in the far-UV ECD spectra changes are linear as a function of the temperature, near-UV ECD data indicate a non-linear and thus, cooperative unfolding mechanism of these proteins. Ensemble of ECD spectra deconvoluted gives both conformational weights and insight to a protein folding↔unfolding mechanism. We found that the Lb293 band is reporting on the 3D-structure compactness. In addition, the pure near-UV ECD spectrum of the unfolded state is described here for the first time. Thus, ECD folding information now validated can be applied with confidence in a large thermal window (5≤T≤85 °C) compared to NMR for studying the unfolding of Trp↔Tyr residue pairs. In conclusion, folding propensities of important proteins (RNA polymerase II, ubiquitin protein ligase, tryptase-inhibitor etc.) can now be analysed with higher confidence.

The “classical” challenge, raised by Emil Fischer as to why one monosaccharide arylhydrazone adopts a cyclic structure but another an acyclic structure, is answered here. The present comprehensive analysis of hexose and hexosamine arylhydrazones, based on 2D NMR spectroscopy and theoretical modeling, has established that the chain of hydrogen bonds needed for conformational selection can only be completed for D-glucosamine derivatives. Thus, D -glucosamine 4-nitrophenyl-hydrazone exclusively adopts its cyclic form, but any configurational changes imply the formation of acyclic structures. In conclusion, three criteria dominate structure selection, namely 1) an amino function at the C-2 position, 2) the “all-equatorial” substitution mode of the pyranoid ring, and 3) an electron- withdrawing group on the arylhydrazone are all needed to get the cyclic form only.

The synthesis, in vitro evaluation and conformational study of several small-size peptides acting as antibacterial agents are reported. Among the compounds evaluated, the peptides Arg-Gln-Ile-Lys-Ile-Trp-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2 , Arg-Gln-Ile-Lys-Ile-Arg-Arg-Met-Lys-Trp-Arg-NH2 , and Arg-Gln-Ile-Trp-Trp-Trp-Trp-Gln-Arg-NH2 exhibited significant antibacterial activity. These were found to be very active antibacterial compounds, considering their small molecular size. In order to better understand the antibacterial activity obtained for these peptides, an exhaustive conformational analysis was performed, using both theoretical calculations and experimental measurements. Molecular dynamics simulations using two different media (water and trifluoroethanol/water) were employed. The results of these theoretical calculations were corroborated by experimental circular dichroism measurements. A brief discussion on the possible mechanism of action of these peptides at molecular level is also presented. Some of the peptides reported here constitute very interesting structures to be used as starting compounds for the design of new small-size peptides possessing antibacterial activity.

The essential involvement of water in most fundamental extra-cellular and intracellular processes of proteins is criticallyreviewed and evaluated in this article. The role of water in protein behavior displays structural ambivalence; it can protectthe disordered peptide-chain by hydration or helps the globular chain-folding, but promotes also the protein aggregation,as well (see: diseases). A variety of amyloid diseases begins as benign protein monomers but develops then into toxic amyloidaggregates of fibrils. Our incomplete knowledge of this process emphasizes the essential need to reveal the principles ofgoverning this oligomerization. To understand the biophysical basis of the simpler in vitro amyloid formation may help todecipher also the in vivo way. Nevertheless, to ignore the central role of the water’s effect among these events means toreceive an uncompleted picture of the true phenomenon. Therefore this review represents a stopgap role, because the mostpublished studies—with a few exceptions—have been neglected the crucial importance of water in the protein research. Thefollowing questions are discussed from the water’s viewpoint: (i) interactions between water and proteins, (ii) proteinhydration/dehydration, (iii) folding of proteins and miniproteins, (iv) peptide/protein oligomerization, and (v) amyloidosis.Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.Keywords: protein hydration; folding; oligomers; amyloid fibrils; miniproteins

Here we present folding associated aggregation propensity of three Trp-cage foldamers: E0, E5 and E10, models of different size but still comparable molecular properties. ECD, VCD and FT-IR spectroscopy measurements were used to monitor their concentration dependent, heat induced (5°C 65°C) alpha-beta fold transition. The ECD curves of E0 are referring to an ensemble of highly dynamic structures. ECD of both E5 and E10 foldamers show the expected Trp-cage fold, dominated by their -helical properties. No sign of -structures was revealed by ECD at any conditions (5°C

Trp-cage miniprotein was used to investigate the role of a salt-bridge (Asp9–Arg16) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side-chains are also part of a dense interaction network composed of electrostatic, H-bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt-bridge with those of the salt-bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp-cage miniproteins is more complex than a simple two-state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt-bridge stabilization; in the miniprotein variants equipped with salt-bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt-bridge deleted mutants. In this molecular framework Arg16 stabilizes more than Asp9 does, because of its higher degree of 3D-fold cooperation. In conclusion, the XxxYyy16 salt-bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays a crucial role in differentiation of oligodendrocytes. Here we first demonstrated by multinuclear NMR that the extended disordered segments are localized at the N- and C-terminals straddling a flexible region. We showed by affinity chromatography, fluorescence spectroscopy and circular dichroism that GTP binds to TPPP/p25 likely within the flexible region; neither positions nor intensities of the peaks in the assigned terminals were affected by GTP. In addition, we demonstrated that TPPP/p25 specifically hydrolyses GTP in an Mg2+-dependent manner. The GTPase activity is comparable with the intrinsic activities of small G proteins suggesting its potential role in multiple physiological processes.

The modular C1r protein is the first protease activated in the classical complement pathway, a key component of innate immunity. Activation of the heteropentameric C1 complex, possibly accompanied by major intersubunit re-arrangements besides proteolytic cleavage, requires targeted regulation of flexibility within the context of the intramolecular and intermolecular interaction networks of the complex. In this study, we prepared the two complement control protein (CCP) modules, CCP1 and CCP2, of C1r in their free form, as well as their tandem-linked construct, CCP1CCP2, to characterize their solution structure, conformational dynamics and cooperativity. The structures derived from NMR signal dispersion and secondary chemical shifts were in good agreement with those obtained by X-ray crystallography. However, successful heterologus expression of both the single CCP1 module and the CCP1CCP2 constructs required the attachment of the preceding N-terminal module, CUB2, which could then be removed to obtain the properly folded proteins. Internal mobility of the modules, especially that of CCP1, exhibited considerable changes accompanied by interfacial chemical shift alterations upon the attachment of the C-terminal CCP2 domain. Our NMR data suggest that in terms of folding, stability and dynamics, CCP1 is heavily dependent on the presence of its neighboring modules in intact C1r. Therefore, CCP1 could be a focal interaction point, capable of transmitting information towards its neighboring modules.

Here we report a thorough analysis of cross-predictions between coiled-coil and disordered protein segments using various prediction algorithms for both sequence classes. Coiled-coils are often predicted to be unstructured, consistent with their obligate multimeric nature, whereas reverse cross-predictions are rare due to the regularity of coiled-coil sequences. We propose the simultaneous use of the programs Coils and IUPred to achieve acceptable prediction accuracy and minimize the extent of cross-predictions. The relevance of observed cross-predictions might be that disordered sequences can adopt coiled-coil conformation relatively easily during protein evolution.

A combined but independently applied NMR and QM procedure has been used to investigate the conformational properties of the exchangeable hydroxyl protons of polyalcohols. In this study, to demonstrate the applicability of such a strategy, we investigated a simple monosaccharide, i.e. α- and β-anomers of a D-glucopyranoside derivative. The redundant set of experimental vicinal homonuclear and heteronuclear scalar couplings involving the OH-protons obtained for both anomers of our model compound were simultaneously analyzed to yield the preferred OH-rotamer populations and moreover to parametrize a new Karplus-type equation for 3JC(i−1)OH(i) coupling. The populations of the lowest energy conformers and the conformational-averaged coupling constants were independently calculated using the QM approach in both vacuum and chloroform. The similarity of the estimated rotamer populations obtained by two very different techniques and the similarity of the experimental and calculated coupling constants suggest that these approaches can be used in conjunction and in a fully integrated way to determine a more accurate atomic level description of molecular conformers. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009

The outline of a universal cell-free translation system capable of site-specific insertion of any types of labeled amino acids is presented. The system could be an invaluable tool for NMR spectroscopy by making the exclusive and exact labeling of the segments of interest possible. Although the development of such a system requires considerable efforts and can not be expected to be available in the next few years, we argue that recent findings concerning the translation apparatus provide clues for overcoming the major difficulties that might arise. We propose a genetic code and a reactor expected to fulfill the specific requirements. Importantly, incomplete systems could also be useful to study selected functional aspects of a number of proteins, examples of which are also given. BioEssays 30:772–780, 2008. © 2008 Wiley Periodicals, Inc.

The activity of calpain is controlled by the free intracellular calcium level and by the protein’s intrinsically disordered endogenous inhibitor, calpastatin, mediated by short conserved segments: subdomains A–C. The exact binding mode of calpastatin to the enzyme has until now been unclear. Our NMR data of the 141 amino acid long inhibitor, with and without calcium and calpain, have revealed structural changes and a tripartite binding mode, in which the disordered inhibitor wraps around, and contacts, the enzyme at three points, facilitated by flexible linkers. This unprecedented binding mode permits a unique combination of specificity, speed and binding strength in regulation.Structured summaryMINT-6549073:Calpain-2 catalytic subunit (uniprotkb:P04632), Calpain-2 catalytic subunit (uniprotkb:P17655) and calpastatin (uniprotkb:P20810) physically interact (MI:0218) by nuclear magnetic resonance (MI:0077)

First principle models of catalytic apparatus of enzymes can be used for studying stability as well as the atomic details of a catalytic mechanism. For example, the catalytic triad of chymotrypsin was recently investigated by using an ab initio geometry optimized (Hudaky and Perczel, Proteins: Struct Funct Genet, 2006, 62, 749) self-stabilizing molecule ensemble without the presence of the complete enzyme and substrate. Several parameters of the above catalytic reaction turned out to be the same within the model and the in vitro enzymatic reaction. Among the numerous parameters of the catalytic process geometrical changes of the catalytic histidine was investigated here and the variation of its pKa value was determined. A relatively large range, 3.5 unit, was determined as the variation of pKa as function of the conformational subspace available in serine proteases. Comparing PDB structures of the free and the complex enzymes it was shown, that histidine, after accepting the proton from the OH group of the catalytic serine, undergoes a minor conformational change accompanied by a 2.5 unit decrease in pKa. We conclude that the changes of pKa during catalysis are predominantly determined by the geometrical arrangement of the histidine moiety and this change serves as a significant driving force in the catalytic process. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

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.

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.

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.

Intrinsic conformational characteristics of β-peptides built up from simple achiral and chiral β-amino acid residues (i.e., HCO-β-Ala-NH2, HCO-β-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-(β-Ala)k-NH2 and HCO-(β-Abu)k-NH2 (1 ≤ k ≤ 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 β-Ala and β-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-(β-HVal-β-HAla-β-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. © 2005 Wiley Periodicals, Inc. J Comput Chem 27: 20–38, 2006

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.

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. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.

Potential energy surfaces of the model peptide HCO-L-Ala-NH2 were calculated using polarizable continuum model (PCM) for the description of aqueous solution at RHF/3-21G, RHF/6-31+G(d), and B3LYP/6-31+G(d) levels of theory. Energy minima were optimized at all three levels as well as at B3LYP/PCM/6-311++G(d,p) level of theory. Results were correlated to experimental data of protein structures retrieved from PDB SELECT. It is concluded that alanine residues of proteins are modeled better by PCM results than by gas-phase calculations on the alanine diamide model (frequently called alanine dipeptide model). The currently available version of the PCM model implemented in Gaussian 03 provides a reasonable alternative to anticipate solvation effects without the computational costs of introducing explicit solvent molecules into the model system. Frequencies calculated at RHF/PCM/6-31+G(d) and B3LYP/PCM/6-31+G(d) levels of theory show high correlation; thus, RHF results have their own merit. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 1522–1531, 2004

The present study focuses on important questions associated with modeling of peptide and protein stability. Computing at different levels of theory (RHF, B3LYP) for a representative ensemble of conformers of di- and tripeptides of alanine, we found that the Gibbs Free Energy values correlate significantly with the total electronic energy of the molecules (0.922or = R2). For noncovalently attached but interacting peptide subunits, such as [For-NH2]2 or [For-L-Ala-NH2]2, we have found, as expected, that the basis set superimposition error (BSSE) is large in magnitude for small basis set but significantly smaller when larger basis sets [e.g., B3LYP/6-311++G(d,p)] are used. Stability of the two hydrogen bonds of antiparallel beta-pleated sheets were quantitatively determined as a function of the molecular structure, S10 and S14, computed as 4.0 +/- 0.5 and 8.1 +/- 1.1 kcal/mol, respectively. Finally, a suitable thermoneutral isodesmic reaction was introduced to scale both covalently and noncovalently attached peptide units onto a common stability scale. We found that a suitable isodesmic reaction can result in the total electronic energy as well as the Gibbs free energy of a molecule, from its "noninteracting" fragments, as accurate as a few tenths of a kcal per mol. The latter observation seems to hold for peptides regardless of their length (1or = nor = 8) or the level of theory applied.

Different protein architectures show strong similarities regardless of their amino acid composition: the backbone folds of the different secondary structural elements exhibit nearly identical geometries. To investigate the principles of folding and stability properties, oligopeptide models (that is, HCO-(NH-L-CHR-CO)(n)-NH(2)) have been studied. Previously, ab initio structure determinations have provided a small amount of information on the conformational building units of di- and tripeptides. A maximum of nine differently folded backbone types is available for any natural alpha-amino acid residue, with the exception of proline. All of these conformers have different relative energies. The present study compiles an ab inito database of optimized HCO-(L-Xxx)(n)-NH(2) structures, where 1

The third helix of the homeodomain of the Antennapedia homeoprotein can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. A 16-amino acid-long peptide fragment, called penetratin, is internalized by the cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, penetratin and two analogs were synthesized:The conformation of penetratin peptides 1-3 was examined in both extracellular matrix-mimetic and membrane-mimetic environments. (1)H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. Peptides 1-3 were labeled by reacting their N-terminal free amino group with fluorescein isothiocyanate (FITC). Membrane translocation of the labelled peptides was studied with cell cultures [WEHI 164 murine fibrosarcoma cells (WC/1); chicken fibroblast cells (CEC-32); chicken monocytic cells (HD-11); human fibroblast (SV 80) and human monocytic cells (MonoMac-6)]. Confocal laser scanning microscopy and flow cytometry assay were used to study membrane translocation. Amphiphilicity was calculated for each peptide. In our experiments all the penetratin peptides penetrated into the cells. Helical conformation and membrane translocation ability showed little correlation: substitution of the two Trp with Phe increased the stability of helical conformation but decreased membrane translocation activity. The results of fluorescence microscopy and flow cytometry show that penetratin can be translocated into the cells by two mechanisms: endocytosis and direct transport through the cell membrane.

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

Knowledge of chemical shift-structure relationships could greatly facilitate the NMR chemical shift assignment and structure refinement processes that occur during peptide/protein structure determination via NMR spectroscopy. To determine whether such correlations exist for polar side chain containing amino acid residues the serine dipeptide model, For-L-Ser-NH(2), was studied. Using the GIAO-RHF/6-31+G(d) and GIAO-RHF/TZ2P levels of theory the NMR chemical shifts of all hydrogen ((1)H(N), (1)H(alpha), (1)H(beta1), (1)H(beta2)), carbon ((13)C(alpha), (13)C(beta), (13)C') and nitrogen ((15)N) atoms have been computed for all 44 stable conformers of For-L-Ser-NH(2). An attempt was made to establish correlation between chemical shift of each nucleus and the major conformational variables (omega(0), phi, psi, omega(1), chi,(1) and chi(2)). At both levels of theory a linear correlation can be observed between (1)H(alpha)/phi, (13)C(alpha)/phi, and (13)C(alpha)/psi. These results indicate that the backbone and side-chain structures of For-L-Ser-NH(2) have a strong influence on its chemical shifts.

At the dawn of the new millenium, new concepts are required for a more profound understanding of protein structures. Together with NMR and X-ray-based 3D-stucture determinations in silico methods are now widely accepted. Homology-based modeling studies, molecular dynamics methods, and quantum mechanical approaches are more commonly used. Despite the steady and exponential increase in computational power, high level ab initio methods will not be in common use for studying the structure and dynamics of large peptides and proteins in the near future. We are presenting here a novel approach, in which low- and medium-level ab initio energy results are scaled, thus extrapolating to a higher level of information. This scaling is of special significance, because we observed previously on molecular properties such as energy, chemical shielding data, etc., determined at a higher theoretical level, do correlate better with experimental data, than those originating from lower theoretical treatments. The Ramachandran surface of an alanine dipeptide now determined at six different levels of theory [RHF and B3LYP 3-21G, 6-31+G(d) and 6-311++G(d,p)] serves as a suitable test. Minima, first-order critical points and partially optimized structures, determined at different levels of theory (SCF, DFT), were completed with high level energy calculations such as MP2, MP4D, and CCSD(T). For the first time three different CCSD(T) sets of energies were determined for all stable B3LYP/6-311++G(d,p) minima of an alanine dipeptide. From the simplest ab initio data (e.g., RHF/3-21G) to more complex results [CCSD(T)/6-311+G(d,p)//B3LYP/6-311++G(d,p)] all data sets were compared, analyzed in a comprehensive manner, and evaluated by means of statistics. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1026–1042, 2003

NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.

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.

Relative stabilities of rotamers of the N-acetyl-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-L-seryl-N'-methyl amide (1) and eleven analogous molecules containing beta-galactose, alpha- and beta-mannose, alpha- and beta-glucose, and L-threonine were calculated to learn whether they could explain the natural preference for 1 in linkages between the carbohydrate and protein in glycoproteins. The lowest energy rotamers of four O-glycoside models of serine diamide were identified with a Monte Carlo search coupled with molecular mechanics (MM2*). These rotamers were further optimized with an ab initio level of theory (HF/6-31G(d)). Subsequently, B3LYP/6-31 + G(d) single point energies were calculated for the most stable HF structures. The most favorable interactions are present in 1 and its glucose analogue. The monosaccharide for the carbohydrate antenna is anchored to the serine residue with an AcNH...O=C-NHMe hydrogen bond in the most stable rotamers. The mannose analogue and the beta-anomers are considerably less stable according to the MM2* and especially to the ab inito energy values. The three analogues have HF/6-31 G(d) energies which are 4-6 kcal mol-1 higher; the single point B3LYP/6-31 + G(d)//HF/6-31 G(d) calculations yield preferences of 3-5 kcal mol-1 for 1. The most stable L-threonine analogues show a behaviour very similarly to the corresponding serine analogues. The ZPE and thermal correction components of the calculated delta H298 and delta G298 values are relatively small (< 0.4 kcal mol-1). However, the T delta S298 term can be as large as 2.6 kcal mol-1. The entropy terms stabilize the alpha-anomers relative to beta-anomers, and ManNAc relative to GalNAc. The largest stabilization effect is observed for one of the rotamers of the alpha-anomer of ManNAc.

The homeodomain, the DNA-binding domain of Antennapedia homeoprotein, is composed of three α-helices and one β-turn between helices II and III. Its third helix from the N-terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix-mimetic and membrane-mimetic environments; 1H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14-Phe)-penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane-mimetic solvent system (TFEd2/water = 9 : 1), on the basis of 553 distance restraints, the 4–12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H-bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix-like conformation. However, while (6,14-Phe)-penetratin displays both α-helical and 310-helical features, the structure of peptide 3 is predominantly a 310-helix. Of the three peptides, surprisingly (6,14-Phe)-penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix-like secondary structures. In a highly aqueous molecular system (TFEd2/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short-range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix-like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.

The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI␣(Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays. All three molecules have a similar fold composed from three antiparallel β-pleated sheets with three disulfide bridges. The proteinase binding loop has a somewhat distinct geometry in all three peptides. Moreover, the stabilization of the structure is different in SGCI and SGTI. Proton–deuterium exchange experiments are indicative of a highly rigid core in SGTI but not in SGCI. We suggest that the observed structural properties play a significant role in the specificity of these inhibitors.

With the construction and implementation of a logical and standardized numbering of atomic nuclei, to define mono-, di-, and oligo-peptide systems, automation of input file generation and data extraction could greatly improve the efficiency of the search for the structural energy minima on the potential energy hypersurface of these systems. The internal hierarchy of the database covering constitutional structures, protective groups, levels of theory, and basis sets used, as well as the variety of possible conformations, is also discussed. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002

Employing introductory (3-21G RHF) and medium-size (6-311CCG B3LYP) ab initio calculations, complete conformational libraries, containing as many as 27 conformers, have been determined for diamide model systems incorporating the amino acids valine (Val) and phenylalanine (Phe). Conformational and energetic properties of these libraries were analyzed. For example, significant correlation was found between relative energies from 6-311CCG B3LYP and single-point B3LYP/6-311CCG//RHF/3-21G calculations. Comparison of populations of molecular conformations of hydrophobic aromatic and nonaromatic residues, based on their ab initio relative energies, with their natural abundance indicates that, at least for the hydrophobic core of proteins, the conformations of Val (Ile, Leu) and Phe (Tyr, Trp) are controlled by the local energetic preferences of the respective amino acids. c 2001 John Wiley & Sons, Inc. J Comput Chem 22: 732-751, 2001

Chemical shielding anisotropy tensors have been determined for all twenty-seven characteristic conformers of For-L-Val-NH2 using the GIAO-RHF formalism with the 6-31+G* and TZ2P basis sets. The individual chemical shifts and their conformational averages have been compared to their experimental counterparts taken from the BioMagnetic Resonance Bank (BMRB). At the highest level of theory applied, for all nuclei but the amide proton, deviations between statistically averaged theoretical and experimental chemical shifts are as low as 1–3 %. Correlated chemical shift plots of selected nuclei, as function of the respective ϕ, ψ, χ1, and χ2 torsional angles, have been generated. On two-dimensional chemical shift–chemical shift plots, for example, 1HNH–15NNH and 15NNH–13Cα, regions corresponding to major conformational clusters have been identified, providing a basis for the quantitative identification of conformers from NMR shift data. Experimental NMR resonances of nuclei of valine residues have been deduced from 18 selected proteins, resulting in 93 1Hα–13Cα chemical shift pairs. These experimental results have been compared to relevant ab initio values revealing remarkable correlation between the two sets of data. Correlations of 1Hα and 13Cα values with backbone conformational parameters (ϕ and ψ) have also been found for all pairs (e.g. 1Hα/ϕ and 13Cα/ϕ) but 1Hα/ψ. Overall, the appealing idea of establishing backbone folding of proteins by employing chemical shift information alone, obtained from selected multiple-pulse NMR experiments (e.g. 2D-HSQC, 2D-HMQC, and 3D-HNCA), has received further support.

Geometric and energetic properties of a diamide of serine, HCO-NH-L-CH(CH2OH)CO-NH2, are investigated by standard methods of computational quantum chemistry. Similarly to other amino acid residues, conformational properties of HCO-L-Ser-NH2 can be derived from the analysis of its ED E(, ;1,2) hypersurface. Reoptimization of 44 RHF/3-21G conformers at the RHF/6-311CCG level resulted in 36 minima. For all conformers, geometrical properties, including variation of H-bond parameters and structural shifts in the torsional space, are thoroughly investigated. Results from further single-point energy calculations at the RHF, DFT, and MP2 levels, performed on the entire conformational data set, form a database of 224 energy values, perhaps the largest set calculated so far for any single amino acid diamide. A comprehensive analysis of this database reveals significant correlation among energies obtained at six levels of ab initio theory. Regression parameters provide an opportunity for extrapolation in order to predict the energy of a conformer at a high level by doing explicit ab initio computations only for a few selected conformers. The computed conformational and relative energy data are compared with structural and occurrence results derived from a nonhomologous

NMR chemical shielding anisotropy tensors have been computed, employing several basis sets and the GIAO-RHF and GIAO-MP2 formalisms of electronic structure theory, for all the atoms of the five and nine typical backbone conformers of For-Gly-NH2 and For-L-Ala-NH2, respectively. Multidimensional chemical shift plots, as a function of the respective backbone fold, have been generated for both peptide models. On the 2D 1 H NH - 15 N NH and 15 N NH - 13 C plots the most notable feature is that at all levels of theory studied the backbone conformers cluster in different regions. Computed chemical shifts, as well as their averages, have been compared to relevant experimental values taken from the BioMagnetic Resonance Bank (BMRB). At the highest levels of theory, for all nuclei but the amide protons, deviations between statistically averaged theoretical and experimental shifts are as low as 1-3%. These results indicate that chemical shift information from selected multiple-pulse NMR experiments (e.g., 2D-HSQC and 3D-HNCA) could directly be employed to extract folding information for polypeptides and proteins. c 2000 John Wiley & Sons, Inc. J Comput Chem 21: 882-900, 2000

In 1968 C. Venkatachalam (Biopolymers, Vol. 6, pp. 1425-1436) predicted the ideal forms of beta-turns (type I, type II, etc.) based entirely on theoretical calculations. Subsequently, over a thousand x-ray structures of different globular proteins have been analyzed, with results suggesting that the most important form among the hairpin conformers is the type I beta-turn. For the latter type of hairpin conformation, the original computations had predicted phi i+I = -60 degrees, psi i+1 = -30 degrees, phi i+2 = -90 degrees, and psi i +2 = 0 degrees as backbone torsion angle values, and these have been used from that time as reference values for the identification of the type I beta-turn. However, it has never been clarified whether these "ideal" backbone torsion angle values exist in real structures, or whether these torsion angles are only "theoretical values." Using the most recent release of the Protein Data Bank (1994), a survey has been made to assign amino acid pairs that approach the ideal form of the type I beta-turn. The analysis resulted in four sequences where the deviation from ideal values for any main-chain torsion angles was less than 2 degrees. In order to determine whether such a backbone fold is possible only in proteins owing to fortuitous cooperation of different folding effects, or whether it occurs even in short peptides, various attempts have been made to design the optimal amino acid sequence. Such a peptide model compound adopting precisely the predicted torsion angle values [phi i+1 = -60 degrees, psi i +1 = -30 degrees, phi i +2 = -90 degrees, and psi i+2 = 0 degrees] could provide valuable information. The solid state conformation of cyclo[(delta)Ava-Gly-Pro-Thr(OtBu)-Gly] reported herein, incorporating the -Pro-Thr- subunit, yields values suggesting that the "ideal" type I beta-turn is even possible for a peptide where there are no major environmental effects present.

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) beta-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of beta-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the beta-turn band showed up between 1639 and 1633 cm-1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band was also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3(10)-helical polypeptides and proteins in D2O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508-512; H. H. Mantsch, A. Perczel, M. Hollosi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch, A. Perczel, M. Hollosi, and G. Fasman (1992), Biopolymers, Vol. 33, pp. 201-207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Todd, and G. L. Millhauser (1992), Nature, Vol. 359, pp. 653-655], suggest that the amide I band, with a major contribution from the acceptor C = O of the 1

The β-turn represents a structural element frequently encountered in globular proteins. However, in spite of various theoretical and experimental studies the ir signature bands of pure β-turns are still not established beyond doubt. Although considerable information exists now on the ir spectra of β-helical and β-sheet structures, the lack of knowledge concerning turn structures in general, and that of β-turns in particular, presents a major uncertainty in the estimation of global protein secondary structures from ir spectroscopic data. To obtain more specific information about the characteristic amide bands in β-turns, we report herein an ir spectroscopic analysis of a series of five cyclic pseudo-hexapeptides known to form β-turns from previous CD and nmr studies [A. Perczel, M. Hollosi, B. M. Foxman, and G. D. Fasman (1991) Journal of the American Chemical Society, Volume 113, pp. 9772-9784 ]. We show here that in these cyclic peptides the amide groups involved in β-turns that comprise a ten-membered hydrogen-bonded ring (and represent the first H-bond pair in a β-sheet), give rise to characteristic amide I bands in the range 1638–1646 cm−1, with the exact position depending on the solvent and the nature of the side-chain substituents. © 1993 John Wiley & Sons, Inc.