Showing papers in "Biopolymers in 2001"

Generalized-ensemble algorithms for molecular simulations of biopolymers.

Abstract: In complex systems with many degrees of freedom such as peptides and proteins, there exists a huge number of local-minimum-energy states. Conventional simulations in the canonical ensemble are of little use, because they tend to get trapped in states of these energy local minima. A simulation in generalized ensemble performs a random walk in potential energy space and can overcome this difficulty. From only one simulation run, one can obtain canonical-ensemble averages of physical quantities as functions of temperature by the single-histogram and/or multiple-histogram reweighting techniques. In this article we review uses of the generalized-ensemble algorithms in biomolecular systems. Three well-known methods, namely, multicanonical algorithm, simulated tempering, and replica-exchange method, are described first. Both Monte Carlo and molecular dynamics versions of the algorithms are given. We then present three new generalized-ensemble algorithms that combine the merits of the above methods. The effectiveness of the methods for molecular simulations in the protein folding problem is tested with short peptide systems. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 60: 96–123, 2001

Control of peptide conformation by the Thorpe-Ingold effect (C alpha-tetrasubstitution).

Abstract: The preferred conformations of peptides heavily based on the currently extensively exploited achiral and chiral α-amino acids with a quaternary α-carbon atom, as determined by conformational energy computations, crystal-state (x-ray diffraction) analyses, and solution (1H-NMR and spectroscopic) investigations, are reviewed. It is concluded that 310/α-helical structures and the fully extended (C5) conformation are preferentially adopted by peptide sequences characterized by this family of amino acids, depending upon overall bulkiness and nature (e.g., whether acyclic or C ↔ C cyclized) of their side chains. The intriguing relationship between α-carbon chirality and bend/helix handedness is also illustrated. γ-Bends and semiextended conformations are rarely observed. Formation of β-sheet structures is prevented. © 2002 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 60: 396–419, 2001

FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage.

Abstract: Articular cartilage, a connective tissue that provides resistance to compressive forces during joint movements, has not been examined in detail by conventional Fourier transform infrared (FTIR) spectroscopy, microspectroscopy (FTIRM), or imaging (FTIRI). The current study reports FTIRM and FTIRI analyses of normal bovine cartilage and identifies the specific molecular components of cartilage that contribute to its IR spectrum. FTIRM data acquired through the superficial, middle, and deep zones of thin sections of bovine articular cartilage showed a variation in intensities of the absorbance bands that arise from the primary nonaqueous components of cartilage, collagen, and proteoglycan (primarily aggrecan) and thus reflected the differences in quantity of these specific components. The spectra of mixtures of model compounds, which had varying proportions of type II collagen and aggrecan, were analyzed to identify spectral markers that could be used to quantitatively analyze these components in cartilage. Collagen and aggrecan were then imaged by FTIRI based on markers found in the model compounds. Polarization experiments were also performed to determine the spatial distribution of the collagen orientation in the different zones of cartilage. This study provides a framework in which complex pathological changes in this heterogeneous tissue can be assessed by IR microscopic imaging.

Electronic properties, hydrogen bonding, stacking, and cation binding of DNA and RNA bases.

Abstract: This review summarizes results concerning molecular interactions of nucleic acid bases as revealed by advanced ab initio quantum chemical (QM) calculations published in last few years. We first explain advantages and limitations of modern QM calculations of nucleobases and provide a brief history of this still rather new field. Then we provide an overview of key electronic properties of standard and selected modified nucleobases, such as their charge distributions, dipole moments, polarizabilities, proton affinities, tautomeric equilibria, and amino group hybridization. Then we continue with hydrogen bonding of nucleobases, by analyzing energetics of standard base pairs, mismatched base pairs, thio-base pairs, and others. After this, the nature of aromatic stacking interactions is explained. Also, nonclassical interactions in nucleic acids such as interstrand bifurcated hydrogen bonds, interstrand close amino group contacts, C—H … O interbase contacts, sugar–base stacking, intrinsically nonplanar base pairs, out-of-plane hydrogen bonds, and amino–acceptor interactions are commented on. Finally, we overview recent calculations on interactions between nucleic acid bases and metal cations. These studies deal with effects of cation binding on the strength of base pairs, analysis of specific differences among cations, such as the difference between zinc and magnesium, the influence of metalation on protonation and tautomeric equlibria of bases, and cation–π interactions involving nucleobases. In this review, we do not provide methodological details, as these can be found in our preceding reviews. The interrelation between advanced QM approaches and classical molecular dynamics simulations is briefly discussed. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 3–31, 2002; DOI 10.1002/bip.10048

Influence of thermal history on the structural and mechanical properties of agarose gels.

Abstract: Using a multitechnique approach, two temperature domains have been identified in agarose gelation Below 35°C, fast gelation results in strong, homogeneous and weakly turbid networks The correlation length, evaluated from the wavelength dependence of the turbidity, is close to values of pore size reported in the literature Above 35°C, gelation is much slower and is associated with the formation of large-scale heterogeneities that can be monitored by a marked change in the wavelength dependence of turbidity and visualised by transmission electron microscopy Curing agarose gels at temperatures above 35°C, and then cooling them to 20°C, produces much weaker gels than those formed directly at 20°C Dramatic reductions in the elastic modulus and failure strain and stress are found in this case as a result of demixing during cure An interpretation, based on the kinetic competition between osmotic forces (in favor of phase separation) and elastic forces (that prevent it) is proposed © 2001 John Wiley & Sons, Inc Biopolymers 59: 131–144, 2001

Methods and strategies of peptide ligation.

Abstract: This review focuses on the concept, methods, and strategies of orthogonal peptide ligation. It updates our previous review in 1999 on the same subject matter in Biopolymers (Peptide Science, 1999, Vol. 51, p. 311). Orthogonal peptide ligation is an amino terminal specific method to couple chemically unprotected peptides or proteins derived from synthetic or biosynthetic sources. Unlike conventional chemical methods, peptide ligation methods do not require coupling reagents or protection schemes, but are achieved through a variable chemoselective capture step and then an invariable intramolecular acyl transfer reaction. It is also a convergent method with the fewest steps. More than a dozen orthogonal ligation methods have been developed based on captures by either imine or thioester chemistries to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. The ligation strategies for multiple segments including sequential and tandem ligations are also discussed.

Gelation behaviour of konjac glucomannan with different molecular weights

Abstract: The deacetylation and gelation of konjac glucomannan (KGM) following alkali addition was investigated by Fourier transform infrared, while the rheological properties of KGM with different molecular weights were studied by dynamic viscoelastic measurements in shear mode and penetration force tests. It was found that gelation occurred after significant deacetylation had taken place. Rheometrical studies revealed that KGM with different molecular weights exhibited different gelation characteristics in small amplitude oscillatory shear flow. For the samples of fractionated KGM with lower molecular weights, a decrease in both the storage shear modulus (G') and loss shear modulus (G") was observed during gelation at temperatures above 75 degrees C. It is suggested that the decrease results from the wall slip between sample and measuring geometry owing to a rapid gelation process with syneresis and/or disentanglement of molecular chains adsorbed on the surface of parallel plates from those located in the bulk. Penetration force tests were employed to confirm the occurrence of slippage and thereby no decreases in rigidity of samples were observed during gelation. For the native KGM samples decreases in G' and G" during gelation were not observed, and it is suggested that this is due to the effect of the higher molecular weight and increased solution viscosity of these samples on the gelation kinetics.

Bovine serum albumin observed by infrared spectrometry. I. Methodology, structural investigation, and water uptake.

Abstract: The results of preliminary infrared (IR) spectrometry experiments on bovine serum albumin (BSA) films are presented. An analysis of spectral variations due to raising the temperature and deuteration of N--H groups leads to the assignment of most IR bands of BSA. From this analysis we furthermore deduce that at 115 degrees C only hydrogen bonds established by NbH groups on the still present H(2)O molecules, which are so strongly bound to the protein that they do not evaporate, are weakened, some of which are broken. These N--H...OH(2) groups represent some 5% of all N--H groups in the dried protein. Spectral changes due to hydration by water vapor are also analyzed and a precise method to measure the water-vapor pressure of the atmosphere surrounding the BSA film, or equivalently the relative humidity, is described. Various procedures to measure the number of H(2)O molecules embedded in BSA are then presented and evaluated. One of them is selected as the best one for proteins, because it matches previous measurements based on gravimetric methods. This procedure is subsequently used in a study that is devoted to the determination of the various hydrogen-bond configurations, or interaction configurations, which are adopted by H(2)O molecules during the various steps of hydration of BSA. This first analysis of hydration spectra allows the completion of the assignment of IR bands. The various spectral components of the amide I band, which are interchanged during the hydration process, cannot be assigned to various secondary structures, as is usually proposed. It suggests that this usual assignment should be used with care, especially by taking into account the state of hydration, when one wishes to obtain structural information from it.

The effect of water content on the ordered/disordered structures in starches.

Abstract: (13)C cross-polarization magic angle spinning NMR has been used to study the ordered and disordered structures of starches with different water contents. The amorphous regions of starch have been shown to produce NMR patterns only if they are in a glassy state, the widths, positions, and areas of the peaks to some extent being dependent on the temperature and the water content of the starch. In the amorphous region, the peaks were all Gaussian in shape, while the peaks in the ordered regions had Lorentz profiles. Water contents in the range 10-50% did not influence the proportion of double helices in the starch. Decreasing the water content to 1-3%, however, resulted in a significant decrease in the proportion of double helices, the effect being greater in B- than in A-type starches. It is suggested that short-range order structures in starches (double helices) are stabilized by becoming part of long-range order structures (crystallites).

Rapid boundary element solvation electrostatics calculations in folding simulations: Successful folding of a 23‐residue peptide

Abstract: Solvation effects play a profound role in the energetics of protein folding. While a continuum dielectric model of solvation may provide a sufficiently accurate estimate of the solvation effects, until now this model was too computationally expensive and unstable for folding simulations. Here we proposed a fast yet accurate and robust implementation of the boundary element solution of the Poisson equation, the REBEL algorithm. Using our earlier double-energy scheme, we included for the first time the mathematically rigorous continuous REBEL solvation term in our Biased Probability Monte Carlo (BPMC) simulations of the peptide folding. The free energy of a 23-residue beta beta alpha-peptide was then globally optimized with and without the solvation electrostatics contribution. An ensemble of beta beta alpha conformations was found at and near the global minimum of the energy function with the REBEL electrostatic solvation term. Much poorer correspondence to the native solution structure was found in the "control" simulations with a traditional method to account for solvation via a distance-dependent dielectric constant. Each simulation took less than 40 h (21 h without electrostatic solvation calculation) on a single Alpha 677 MHz CPU and involved more than 40,000 solvation energy evaluations. This work demonstrates for the first time that such a simulation can be performed in a realistic time frame. The proposed procedure may eliminate the energy evaluation accuracy bottleneck in folding simulations.

Distinguishing malignant from normal oral tissues using FTIR fiber-optic techniques.

Abstract: Oral tissue samples were studied using mid-IR fiber-optic attenuated total reflectance spectroscopy and other spectral techniques. The 1745 cm(-1) band, which is assigned to the ester group (C==O) vibration of triglycerides, is a reliable marker that is present in normal tissues but absent or a weak band in malignant oral tissues. Other bands such as C--H stretching bands and the amide bands are also helpful in distinguishing malignant tissues from normal tissues. Subtraction spectra confirmed the above conclusion. In addition, Raman spectroscopic measurements were in agreement with the results observed from FTIR spectra.

Solution structure of native proteins with irregular folds from Raman optical activity

Abstract: Raman optical activity (ROA) spectra have been measured for the proteins hen phosvitin, yeast invertase, bovine alpha-casein, soybean Bowman-Birk protease inhibitor, and rabbit Cd(7)-metallothionein, all of which have irregular folds in the native state. The results show that ROA is able to distinguish between two types of disorder. Specifically, invertase, alpha-casein, the Bowman-Birk inhibitor, and metallothionein appear to possess a "static" type of disorder similar to that in disordered states of poly(L-lysine) and poly(L-glutamic acid); whereas phosvitin appears to possess a more "dynamic" type of disorder similar to that in reduced (unfolded) lysozyme and ribonuclease A and also in molten globule protein states. In the delimiting cases, static disorder corresponds to that found in loops and turns within native proteins with well-defined tertiary folds that contain sequences of residues with fixed but nonrepetitive phi,psi angles; and dynamic disorder corresponds to that envisaged for the model random coil in which there is a distribution of Ramachandran phi,psi angles for each amino acid residue, giving rise to an ensemble of interconverting conformers. In both cases there is a propensity for the phi,psi angles to correspond to the alpha, beta and poly(L-proline) II (PPII) regions of the Ramachandran surface, as in native proteins with well-defined tertiary folds. Our results suggest that, with the exception of invertase and metallothionein, an important conformational element present in the polypeptide and protein states supporting the static type of disorder is that of the PPII helix. Long sequences of relatively unconstrained PPII helix, as in alpha-casein, may impart a plastic (rheomorphic) character to the structure.

Amyloidogenicity and cytotoxicity of islet amyloid polypeptide

Abstract: Insoluble amyloid formation by islet amyloid polypeptide (IAPP) in the islets of Langerhans of the pancreas is a major pathophysiological feature of noninsulin dependent diabetes mellitus (NIDDM) or type II diabetes. Because in vivo formed amyloid colocalizes with areas of cell degeneration and IAPP amyloid aggregates are cytotoxic per se, the process of IAPP amyloid formation has been strongly associated with the progressive pancreatic cell degeneration and thus much of the pathology of type II diabetes. IAPP is a pancreatic polypeptide of 37 residues that, in its soluble form, is believed to play a role as a regulator of glucose homeostasis. The molecular cause and mechanism of the conversion of soluble IAPP into insoluble amyloid aggregates in vivo and its role in disease progress still remain to be clarified. Nevertheless, in the past few years significant progress has been made in understanding the amyloidogenesis pathway of IAPP in vitro and gaining insight into the structural and conformational "requirements" of IAPP amyloidogenesis and related cytotoxic effects. Importantly, several of the studies have revealed significant similarities of the above features of IAPP to other amyloidogenic polypeptides such as the beta-amyloid polypeptide Abeta. This suggests that, at the molecular level, amyloidogenesis, and possibly related cell degeneration and disease pathogenesis by completely different polypeptide sequences, may obey to common structural and conformational "rules" and follow similar molecular pathways. This review describes studies on the structural and conformational features of IAPP amyloid formation and cytotoxicity, and the application of the obtained knowledge for the understanding of the molecular mechanism of the IAPP amyloidogenesis pathway and the related cytotoxicity.

Prediction of the subcellular location of prokaryotic proteins based on a new representation of the amino acid composition.

Abstract: A new representation of protein sequence is devoted in this paper, in which each protein can be represented by a 20-dimensional (20D) vector of unit length. Inspired by the principle of superposition of state in quantum mechanics, the squares of the 20 components of the vector correspond to the amino acid composition. Using the new representation of the primary sequence and Bayes Discriminant Algorithm, the subcellular location of prokaryotic proteins was predicted. The overall predictive accuracy in the jackknife test can be 3% higher than the result of using amino acid composition directly for the database of sequence identity is less than 90%, but 5% higher when sequence identity is less than 80%. The higher predictive accuracy indicates that the current measure of extracting the information from the primary sequence is efficient. Since the subcellular location restricting a protein's possible function, the present method should also be a useful measure for the systematic analysis of genome data. The program used in this paper is available on request. © 2001 John Wiley & Sons, Inc. Biopolymers 58: 491–499, 2001

A rheological study of the order-disorder conformational transition of xanthan gum.

Abstract: The rheological properties of a moderately concentrated solution of xanthan gum in both the ordered and the disordered state have been studied. Oscillatory shear, steady shear flow, and extensional flow experiments have been performed at different temperatures, covering the order-disorder transition determined by differential scanning calorimetry (DSC). The principle of time/temperature superposition was applied to the xanthan solutions for the different types of flow. Although a master curve covering six decades of frequency could be obtained for the storage modulus over the entire investigated temperature range, less agreement was found for the other modulus. This indicates that the order-disorder transition reflects changes on the molecular scale and slight modification of the physical network structure. To the authors' knowledge, this is the first time that this transition has been observed using these different rheological techniques.

Site-specific examination of secondary structure and orientation determination in membrane proteins: the peptidic (13)C=(18)O group as a novel infrared probe.

Abstract: Detailed site-specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1-(13)C=(18)O labels have been shown to relay site-specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of (13)C and the lack of baseline resolution between the main amide I band and the isotope-edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1-(13)C=(18)O label. The double-isotope label virtually eliminates any contribution from natural abundance (13)C. More importantly, the isotope-edited peak is further red-shifted (in accordance with ab initio Hartree-Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1-(13)C=(18)O is far preferable as a label in comparison to 1-(13)C=(18)O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site-directed dichroism to the structural elucidation of polytopic membrane proteins.

Modeling salt-mediated electrostatics of macromolecules: The discrete surface charge optimization algorithm and its application to the nucleosome

Abstract: Much progress has been achieved on quantitative assessment of electrostatic interactions on the all-atom level by molecular mechanics and dynamics, as well as on the macroscopic level by models of continuum solvation. Bridging of the two representations-an area of active research-is necessary for studying integrated functions of large systems of biological importance. Following perspectives of both discrete (N-body) interaction and continuum solvation, we present a new algorithm, DiSCO (Discrete Surface Charge Optimization), for economically describing the electrostatic field predicted by Poisson-Boltzmann theory using a discrete set of Debye-Huckel charges distributed on a virtual surface enclosing the macromolecule. The procedure in DiSCO relies on the linear behavior of the Poisson-Boltzmann equation in the far zone; thus contributions from a number of molecules may be superimposed, and the electrostatic potential, or equivalently the electrostatic field, may be quickly and efficiently approximated by the summation of contributions from the set of charges. The desired accuracy of this approximation is achieved by minimizing the difference between the Poisson-Boltzmann electrostatic field and that produced by the linearized Debye-Huckel approximation using our truncated Newton optimization package. DiSCO is applied here to describe the salt-dependent electrostatic environment of the nucleosome core particle in terms of several hundred surface charges. This representation forms the basis for modeling-by dynamic simulations (or Monte Carlo)-the folding of chromatin. DiSCO can be applied more generally to many macromolecular systems whose size and complexity warrant a model resolution between the all-atom and macroscopic levels.

Structure of Bombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and (13)C cross-polarization/magic angle spinning NMR.

Abstract: The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated beta-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)(15) with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)(5) and (X-Gly)(15) where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the (13)C CP/MAS NMR result, where the conformation is mainly random coil.

Universal similarity measure for comparing protein structures.

Abstract: We introduce a new variant of the root mean square distance (RMSD) for comparing protein structures whose range of values is independent of protein size. This new dimensionless measure (relative RMSD, or RRMSD) is zero between identical structures and one between structures that are as globally dissimilar as an average pair of random polypeptides of respective sizes. The RRMSD probability distribution between random polypeptides converges to a universal curve as the chain length increases. The correlation coefficients between aligned random structures are computed as a function of polypeptide size showing two characteristic lengths of 4.7 and 37 residues. These lengths mark the separation between phases of different structural order between native protein fragments. The implications for threading are discussed.

Controls exerted by the Aib residue: Helix formation and helix reversal

Abstract: The helix forming properties of the achiral α-amino isobutyric residue (Aib) have been demonstrated by numerous crystal structure analyses of designed and naturally occurring peptides containing one or more Aib residues in the sequence. Experimental and computational results concerning the type of helix obtained, whether the 310-helix with 4 1 type hydrogen bonds or the α-helix with 5 1 hydrogen bonds or mixtures of the two, have been published. This paper deals with residues that, if inserted into a sequence, could perturb the helix-forming propensity afforded by the presence of Aib residues. Examples of structures will be presented in which Pro, Hyp, Gly–Gly, d-Ala–Gly, and Lac have been centrally placed in the sequence. In addition to the formation of helices, detailed experimentally obtained conformation information is presented for the role of the Aib residue in reversing the sense of the helix (the Schellman motif) with the consequent formation of the 6 1 type hydrogen bond or a solvated 6 1 hydrogen bond. Data are presented for 13 molecules with helix reversals at the C-terminus or near the center of the sequence. Published 2002 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 60: 351–365, 2001

Comparative rheological behavior of hyaluronan from bacterial and animal sources with cross‐linked hyaluronan (hylan) in aqueous solution

Abstract: Using a variety of rheological techniques, the behavior of hyaluronan (Mw 0.8-2.2 × 106), cross-linked hyaluronan (hylan) (Mw 1.8-12.5 × 106), and Healon (Mw 5 × 106) (a proprietary hyaluronan) was studied over a large range of molecular weights. The object was to study the effect of the cross-links in hylan on the various rheological parameters, in comparison with linear hyaluronan. There are significant differences. The Huggins constant and the critical overlap parameter C*[ ] are considerably lower for hylan and an increase in moduli at low frequencies was observed for hylan compared with the hyaluronan samples at all molecular weights studied. The results point to a difference in structure in dilute solution for hylan due to the ability to form networks, which can be removed by pressure filtration. In contrast, we do not find an increase of the steady shear viscosity and elastic modulus at higher concentrations when a homogeneous entangled network is reached. We attribute this behavior to the semirigid character of the hyaluronan chain and to the predominance of entanglements over the cross-link points present in hylan in the semidilute domain. Due to the higher apparent molecular weights that are possible with hylan structures but not with the hyaluronans currently available, a wider range of applications can be achieved with hylans when viscoelasticity is required, particularly for the viscosupplementation of synovial fluid damaged by osteoarthritis. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 191-204, 2001

The binding database: Overview and user's guide

Abstract: The large and growing body of experimental data on molecular binding is of enormous value in biology, pharmacology, and chemistry Applications include the assignment of function to biomolecules, drug discovery, molecular modeling, and nanotechnology However, binding data are difficult to find and access because they are available almost exclusively through scientific journals BindingDB, a public, web-accessible database of measured binding affinities, is designed to address this problem BindingDB collects data for natural and modified biomolecules and for synthetic compounds, and provides detailed experimental information Currently, measurements by isothermal titration calorimetry are fully supported; measurements by enzyme inhibition will soon be included as well The web site allows data to be searched by a range of criteria, including binding thermodynamics, sequence homology, and chemical structure, substructure, and similarity Experimentalists are encouraged to publicize their data by entering it into BindingDB via the online forms Such data can be updated or revised by the depositor, if necessary, and will remain publicly accessible User involvement and feedback are welcomed

Effects of structural modifications on some physical characteristics of exopolysaccharides from Lactococcus lactis.

Abstract: The relationship between the primary structure and the chain stiffness of exopolysaccharides (EPSs) and modified EPSs produced by two strains of Lactococcus lactis subsp. cremoris was investigated. The molar mass and radius of gyration of these exopolysaccharides were analyzed by multiangle static light scattering after size-exclusion chromatography. From these results and the chemical structure of the repeating units of the investigated EPSs, the Kuhn lengths could be calculated. We found that the initial Kuhn lengths of the two native EPSs are similar. Modification of the EPSs by removing parts of the side groups resulted in a decrease in both the absolute value and the normalized value of the Kuhn length. It is therefore concluded that partial removal of the side groups of these polysaccharides could make them less efficient as thickeners if no specific interaction with other components occurs.

Phosphoryltyrosyl mimetics in the design of peptide-based signal transduction inhibitors.

Abstract: The central roles played by protein–tyrosine kinase (PTK)-dependent signal transduction in normal cellular regulation and homeostasis have made inappropriate or aberrant functions of certain of these pathways contributing factors to a variety of diseases, including several cancers. For this reason, development of PTK signaling inhibitors has evolved into an important approach toward new therapeutics. Since in these pathways phosphotyrosyl (pTyr) residues provide unique and defining functions either by their creation under the catalysis of PTKs, their recognition and binding by protein modules such as SH2 and phosphotyrosyl binding (PTB) domains, or their destruction by protein–tyrosine phosphatases, pTyr mimetics provide useful general starting points for inhibitor design. Important considerations in the development of such pTyr mimetics include enzymatic stability (particularly toward PTPs), high affinity recognition by target pTyr binding proteins, and good cellular bioavailability. Although small molecule, nonpeptide inhibitors may be ultimate objectives of inhibitor development, peptides frequently serve as display platforms for pTyr mimetics, which afford useful and conceptually straightforward starting points in the development process. Reported herein is a limited overview of pTyr mimetic development as it relates to peptide-based agents. Of particular interest are recent findings that highlight potential limitations of peptides as display platforms for the identification of small molecule leads. One conclusion that results from this work is that while peptide-based approaches toward small molecule inhibitor design are often intellectually satisfying from a structure-based perspective, extrapolation of negative findings to small molecule, nonpeptide contexts should be undertaken with extreme caution. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 60: 32–44, 2001

Ab initio conformational analysis of nucleic acid components: Intrinsic energetic contributions to nucleic acid structure and dynamics

Abstract: In recent years, the use of high-level ab initio calculations has allowed for the intrinsic conformational properties of nucleic acid building blocks to be revisited. This has provided new insights into the intrinsic conformational energetics of these compounds and its relationship to nucleic acids structure and dynamics. In this article we review recent developments and present new results. New data include comparison of various levels of theory on conformational properties of nucleic acid building blocks, calculations on the abasic sugar, known to occur in vivo in DNA, on the TA conformation of DNA observed in the complex with the TATA box binding protein, and on inosine. Tests of the Hartree–Fock (HF), second-order Moller–Plesset (MP2), and Density Functional Theory/Becke3, Lee, Yang and Par (DFT/B3LYP) levels of theory show the overall shape of backbone torsional energy profiles (for γ, e, and χ) to be similar for the different levels, though some systematic differences are identified between the MP2 and DFT/B3LYP profiles. The east pseudorotation energy barrier in deoxyribonucleosides is also sensitive to the level of theory, with the HF and DFT/B3LYP east barriers being significantly lower (∼2.5 kcal/mol) than the MP2 counterpart (∼4.0 kcal/mol). Additional calculations at various levels of theory suggest that the east barrier in deoxyribonucleosides is between 3.0 and 4.0 kcal/mol. In the abasic sugar, the west pseudorotation energy barrier is found to be slightly lower than the east barrier and the south pucker is favored more than in standard nucleosides. Results on the TA conformation suggest that, at the nucleoside level, this conformation is significantly destabilized relative to the global energy minimum, or relative to the A- and B-DNA conformations. Deoxyribocytosine would destabilize the TA conformation more than other bases relative to the A-DNA conformation, but not relative to the B-DNA conformation. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 61–76, 2002; DOI 10.1002/bip.10047

Disparate ionic-strength dependencies of on and off rates in protein-protein association.

Abstract: Electrostatic interactions have been observed to play important roles in the kinetics of protein–protein association. Ionic strength, by its ability to modulate the magnitude of electrostatic interactions, has often been conveniently used to test their presence. From experiments on a wide range of associating proteins, a common feature has emerged: the on rates show strong dependence on ionic strength whereas the off rates are relatively insensitive. Here this feature is explained by an explicit description of a transition state for the association process and the suggestion that this transition is near the final bound state of two proteins. The molecular basis of the transition state in the bimolecular process lies in the fact that the bound state is characterized by local specific (e.g., van der Waals, hydrophobic, and electrostatic) interactions, whereas the unbound state is characterized by translational and rotational freedom. In the transition state the protein–protein pair encounters a free-energy maximum since its translational-rotational entropy is reduced while the specific interactions are not yet attained. In this formalism of the protein–protein association process, the enhancement of on rates by long-range electrostatic interactions can be written (analogous to an ordinary transition-state theory) in the form kon = kexp(−G/kBT), where G is the electrostatic free energy of the transition state. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 427–433, 2001

The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan.

Abstract: Structural characteristics of pectic substances extracted from soybean meal cell walls (water unextractable solids) with a chelating agent-containing buffer (0.05M 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and 0.05M NH(4)-oxalate in 0.05M NaOAc buffer) were studied. The arabinogalactans present as side chains to the rhamnogalacturonan backbone were largely removed by enzymatic hydrolysis using endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining pectic backbone appeared to be resistant to enzymatic degradation by pectolytic enzymes. After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these populations showed that the pectic substances in the original extract contain both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan is absent.

Designing cell-permeant phosphopeptides to modulate intracellular signaling pathways.

Abstract: A central theme in intracellular signaling is the regulatable interaction of proteins via the binding of specialized domains on one protein to short linear sequences on other molecules. The capability of these short sequences to mediate the required specificity and affinity for signal transduction allows for the rational design of peptide-based modulators of specific protein-protein interactions. Such inhibitors are valuable tools for elucidating the role of these interactions in cellular physiology and in targeting such interactions for potential therapeutic intervention. This approach is exemplified by the study of the role of phosphorylation of specific sites on signaling proteins. However, the difficulty of introducing large hydrophilic molecules such as phosphopeptides into cells has been a major drawback in this area. This review describes the application of recently developed cell-permeant peptide vectors in the introduction of biologically active peptides into cells, with particular emphasis on the antennapedia/penetratin, TAT, and signal-peptide based sequences. In addition, the modification of such peptides to increase uptake efficiency and affinity for their targets is discussed. Finally, the use of cell-permeant phosphopeptides to both inhibit and stimulate intracellular signaling mechanisms is described, by reference to the PLCgamma, Grb2, and PI-3 kinase pathways.

Variability of cork from Portuguese Quercus suber studied by solid‐state 13C‐NMR and FTIR spectroscopies

Abstract: A new approach is presented for the study of the variability of Portuguese reproduction cork using solid-state 13C-NMR spectroscopy and photoacoustic (PAS) FTIR (FTIR-PAS) spectroscopy combined with chemometrics. Cork samples were collected from 12 different geographical sites, and their 13C-cross-polarization with magic angle spinning (CP/MAS) and FTIR spectra were registered. A large spectral variability among the cork samples was detected by principal component analysis and found to relate to the suberin and carbohydrate contents. This variability was independent of the sample geographical origin but significantly dependent on the cork quality, thus enabling the distinction of cork samples according to the latter property. The suberin content of the cork samples was predicted using multivariate regression models based on the 13C-NMR and FTIR spectra of the samples as reported previously. Finally, the relationship between the variability of the 13C-CP/MAS spectra with that of the FTIR-PAS spectra was studied by outer product analysis. This type of multivariate analysis enabled a clear correlation to be established between the peaks assigned to suberin and carbohydrate in the FTIR spectrum and those appearing in the 13C-CP/MAS spectra. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 268–277, 2001