Formation of silk fibroin matrices with different texture and its cellular response to normal human keratinocytes

Three forms of silk fibroin (SF) matrices, woven (microfiber), non-woven (nanofiber), and film form, were used to perform a conformational analysis and cell culture using normal human oral keratinocytes (NHOK). To obtain the SF microfiber (SF-M) matrix, natural grey silk was degummed, while the SF film (SF-F) and nanofiber (SF-N) matrices were prepared by casting and electrospinning the formic acid solutions of the regenerated SF, respectively. For insolubilization, as-prepared SF-F and SF-N matrices were chemically treated with an aqueous methanol solution of 50%. The conformational structures of as-prepared and chemically treated SF matrices were investigated using attenuated total reflectance infrared spectroscopy (ATR-IR) and solid-state ¹³C CP/MAS nuclear magnetic resonance (NMR) spectroscopy. The as-cast SF-F matrix formed a mainly β-sheet structure that was similar to the SF-M matrix, whereas the as-spun SF-N matrix had a random coil conformation as the predominant secondary structure. Conformational transitions from random coil to β-sheet of the as-spun SF-N occurred rapidly within 10 min following aqueous methanol treatment, and were confirmed by solid-state ¹³C NMR analysis. To assess the cytocompatibility and cells behavior on the different textures of SF, we examined the cell attachment and spreading of NHOK that was seeded onto the SF matrices, as well as the interaction between the cells and SF matrices. Our results indicate that the SF nanofiber matrix may be more preferable than SF film and SF microfiber matrices for biomedical applications, such as wound dressings and scaffolds for tissue engineering.     

Protein flexibility: multiple molecular dynamics simulations of insulin chain B

Multiple molecular dynamics simulations totaling more than 100 ns were performed on chain B of insulin in explicit solvent at 300 K and 400 K. Despite some individual variations, a comparison of the protein dynamics of each simulation showed similar trends and most structures were consistent with NMR experimental values, even at the elevated temperature. The importance of packing interactions in determining the conformational transitions of the protein was observed, sometimes resulting in conformations induced by localized hydrophobic interactions. The high temperature simulation generated a more diverse range of structures with similar elements of secondary structure and populated conformations to the simulations at room temperature. A broad sampling of the conformational space of insulin chain B illustrated a wide range of conformational states with many transitions at room temperature in addition to the conformational states observed experimentally. The T-state conformation associated with insulin activity was consistently present and a possible mechanism of behavior was suggested.     

Influence of the membrane surface on glycolipid conformation and dynamics. An interpretation of NMR results using conformational energy calculations.

Glycolipids constitute an important class of biomolecules that are involved in biomolecular recognition. The importance of carbohydrate head group conformation in such processes is well recognized. Glycolipids typically occur as minor components of the complex heterogeneous matrix of a biological membrane. As a result, the membrane surface may not only influence head group conformation but also serves as a spatial frame in which the glycolipid is oriented and recognized. In this study, conformational energy calculations have been used to assess the conformational space available to the glucose head group of 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in a liquid-crystalline membrane matrix. 2H NMR quadrupolar splittings are calculated and compared with those observed experimentally. This study demonstrates the importance of including surface interactions when considering the conformational space accessible to cell surface carbohydrates. The empirical approach taken here provides considerable insight at the molecular level, and offers the possibility of exploring even more complex systems.     

Chiroptical methods in conformational analysis

CD spectra of flexible organic molecules in solution are normally very sensitive to conformation. In a system composed of two or more chromophores joined by one or more single bonds and with one fixed or strongly preferred conformation, it is often possible to reproduce the CD spectrum by semiempirical calculations based on interactions between the electronic transitions in the respective chromophores. In a system containing two or more conformations of appreciable weight, the observed CD spectrum is the superposition of those of the individual conformations and has to be analyzed in terms of the individual calculated spectra and the relative energies of the respective conformations. The effect of temperature variation on conformational equilibria and on the composite CD spectra will be discussed and exemplified. © 1995 Wiley-Liss, Inc.     

An amino acid substitution matrix for protein conformation identification

Amino acid substitution matrices play an essential role in protein sequence alignment, a fundamental task in bioinformatics. Most widely used matrices, such as PAM matrices derived from homologous sequences and BLOSUM matrices derived from aligned segments of PROSITE, did not integrate conformation information in their construction. There are a few structure-based matrices, which are derived from limited data of structure alignment. Using databases PDB_SELECT and DSSP, we create a database of sequence-conformation blocks which explicitly represent sequence-structure relationship. Members in a block are identical in conformation and are highly similar in sequence. From this block database, we derive a conformation-specific amino acid substitution matrix CBSM60. The matrix shows an improved performance in conformational segment search and homolog detection.     

Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three-domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C-terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns-length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure-based inhibitor design.     

Effects of chain unsaturation on the equation of state for lipid monolayers at the air-water interface.

An equation of state for lipid monolayers at the air-water interface is presented, which takes into account the effects of the conformation and the number and position of double bonds of the hydrocarbon chains. The total Hamiltonian of the monolayer is assumed to consist of three terms. Two of them are calculated exactly within the limitations of the formulation. These are the two-dimensional entropy of mixing of the lipid and water molecules at the surface and the conformational entropy of the chains using a model available from the literature. These two terms give rise to positive surface pressure. The third term, which includes all energies that are not amenable to calculation, was obtained as the difference between the sum of the two calculated terms and experimental data and is found to represent an approximately area-independent tension. The effects of chain unsaturation on the equation of state were modeled in the present theory in two ways; the chain bend caused by cis double bonds increases the minimal molecular area, and the double bond linkage on a chain decreases the degrees of freedom of the chain. Calculations revealed that the former is highly significant whereas the latter is negligible. The deduced equation of state reproduces experimental data with appropriate values for three parameters, which represent the collapse area, the overlap of adjacent chains, and the combined effects of the intra- and intermolecular interactions other than the surface mixing entropy and the chain conformational energy.     

Limited conformational space for early-stage protein folding simulation

MOTIVATION: The problem of early-stage protein folding is critical for protein structure prediction. The model presented introduces a common definition of protein structures which may be treated as the possible in silico early-stage form of the polypeptide chain. Limitation of the conformational space to the ellipse path on the Ramachandran map was tested as a possible sub-space to represent the early-stage structure for simulation of protein folding. The proposed conformational sub-space was developed on the basis of the backbone conformation, with side-chain interactions excluded. RESULTS: The ellipse-path-limited conformation of BPTI was created using the criterion of shortest distance between Phi, Psi angles in native form of protein and the Phi, Psi angles belonging to the ellipse. No knots were observed in the structure created according to ellipse-path conformational sub-space. The energy minimization procedure applied to ellipse-path derived conformation directed structural changes toward the native form of the protein with SS-bonds system introduced to the procedure. AVAILABILITY: Program 'Ellipse' to create the ellipse-path derived structure available on request: myroterm@cyf-kr.edu.pl     

Conformational changes in DNA and soft modes

In this paper we explore the applicability of the soft mode approach to study the conformational transitions of DNA. It is believed that the A-B conformation change is a first order transition. Soft mode theories only apply to the initial stages of a first order transition. However the mode softening in such a case can be the initiating factor which ultimately leads to the transition. The first order transition is, then, a breakdown of what otherwise would have been a true second order transition. The mode softening is causally connected to onset of the transition. We use the eigenvectors obtained from lattice dynamics calculations to identify the softmode. We use the eigenvector projections to form a force constant matrix that is required to drive a mode soft. We explore the methods by which this force constant matrix can be formed. We suggest that the breaking of specific "water bridges" between phosphate groups in the two single strands can drive the conformation change.     

Toward an accurate theoretical framework for describing ensembles for proteins under strongly denaturing conditions

Our focus is on an appropriate theoretical framework for describing highly denatured proteins. In high concentrations of denaturants, proteins behave like polymers in a good solvent and ensembles for denatured proteins can be modeled by ignoring all interactions except excluded volume (EV) effects. To assay conformational preferences of highly denatured proteins, we quantify a variety of properties for EV-limit ensembles of 23 two-state proteins. We find that modeled denatured proteins can be best described as follows. Average shapes are consistent with prolate ellipsoids. Ensembles are characterized by large correlated fluctuations. Sequence-specific conformational preferences are restricted to local length scales that span five to nine residues. Beyond local length scales, chain properties follow well-defined power laws that are expected for generic polymers in the EV limit. The average available volume is filled inefficiently, and cavities of all sizes are found within the interiors of denatured proteins. All properties characterized from simulated ensembles match predictions from rigorous field theories. We use our results to resolve between conflicting proposals for structure in ensembles for highly denatured states.     

Conformational Changes in DNA and Soft Modes

Abstract

In this paper we explore the applicability of the soft mode approach to study the conformational transitions of DNA. It is believed that the A-B conformation change is a first order transition. Soft mode theories only apply to the initial stages of a first order transition. However the mode softening in such a case can be the initiating factor which ultimately leads to the transition. The first order transition is, then, a breakdown of what otherwise would have been a true second order transition. The mode softening is causally connected to onset of the transition. We use the eigenvectors obtained from lattice dynamics calculations to identify the softmode. We use the eigenvector projections to form a force constimi matrix that is required to drive a mode soft. We explore the methods by which this force constant matrix can be formed. We suggest that the breaking of specific “water bridges” between phosphate groups in the two single strands can drive the conformation change.     

Transitions of the left poly-L-proline II helix-right alpha helix type in the regions of myosin subfragment S2 of cross-striated muscle as a possible conformational basis for a mechanical-chemical act]

Local conformational states of fibrous fragments of myosin molecules from striated muscle have been studied. Analysis of the amino acid sequences of the rat embryonic skeletal muscle myosin heavy chain and the nematode myosin heavy chain have been performed with the aim to estimate the influence of electrostatic interactions on secondary structure stability of these fragments. The heterogeneity of stability of alpha-helical conformation along the fibrous fragment have been found on the basis of estimation of interaction between side group charges and between side group charges and main chain charges. Periodically located short sections have been found in the N-terminal half of the myosin rod where clusters of Asp and Glu destabilize alpha-helical structure being ionized. Changes of the distribution of charges near the latter sections bring about conformational transitions from left-handed polyproline II helix to right alpha-helix or vice versa. The new scheme of orientation of fibrous part of the cross-bridge in relation to the thick filament for various stages of muscle contraction process suggests asynchronous character of transitions in the different sites. It may be proposed that existence of alteration left- and right-helical fragments in N-terminal half of fibrous part of heavy myosin chain determines zigzag form of this part of myosin molecule in resting muscle. A model of the cross-bridge movement in the course of ATP hydrolysis has been suggested.     

Multiple solution conformations and internal rotations of the decapeptide gramicidin S.

The conformations of every C alpha H-C beta H2 moiety of the peptide gramicidin S are reported. Internal rotation occurs, but distinct preferences for one side chain rotamer, greater than 80%, are found for the D-phenylalanine and ornithine residues. Leucine and valine exhibit more extensive averaging while proline is shown to be at least 90% in the Ramachandran B conformation. The data are consistent with the coexistence of many tertiary conformations of gramicidin S; the statistical weights of the twelve major tertiary conformations consistent with the rotamer populations are reported. The relative statistical weights of the tertiary conformers depend upon temperature and solvent. A comparison of the conclusions from this publication and conformations derived by energy minimization procedures is made. Partial agreement was found, but the calculations have not yet predicted the wealth of coexisting tertiary conformations nor accounted for the subtle effects of solvent. It is proposed that a more complete picture of the conformational dynamics of gramicidin S and other peptides will result from calculations which use as a basis the extensive data reported here.     

Correlation between computed conformational properties of cytochrome c peptides and their antigenicity in a T-lymphocyte proliferation assay

By means of conformational energy calculations, we previously showed that the antigenic strength of a series of oligopeptides (derived from the carboxyl terminal sequence of cytochrome c) in a T-lymphocyte proliferation assay depends on their ability to adopt the α-helix conformation. Using experimentally determined statistical weights (within the framework of the Zimm–Bragg theory for the helix–coil transition), here we present a simple free energy analysis of the ability of these peptides to adopt the α-helix conformation in water. The experimental statistical weights have been modified to include the effect of long-range charge–dipole interactions on helix stability. We find that there is a close correlation between the tendency of a peptide to adopt the α-helix conformation and its ability to stimulate antigen-primed T cells. The shortest peptide with a tendency to adopt the α-helix conformation is also the shortest one that exhibits antigenic activity. The rapid and simple method presented here can thus be used to predict relative antigenicities for different peptides derived from cytochrome c.     

All-valence-shell molecular orbital calculations on the chiroptical properties of substituted and unsubstituted 2,5-diketopiperazine structures

The chiroptical properties of L -3-methyl-2,5-diketopiperazine (L -alanylglycyl anhydride) are examined on a theoretical model in which the electronic wave functions are obtained from semi-empirical all-valence-shell molecular orbital calculations. The INDO molecular orbital model is used to perform SCF-MO calculations on the ground states of six conformation isomers of L -3-methyl-2,5-diketopiperazine and two chiral conformational isomers of unsubstituted 2,5-diketopiperazine. Excited-state wave functions are constructed in the virtual orbital-configuration interaction approximation. The rotatory strengths, dipole strengths, oscillator strengths, and dissymmetry factors of the first eight singlet–singlet transitions for each of the eight structures are calculated and reported. Additionally, ground-state dipole moments, net atomic charges, and the first four ionization potentials (calculated according to Koopman's theorem) are computed for each structure. The signs and the magnitudes of the rotatory strengths are found to be extremely sensitive to the conformation of the piperazine ring as well as to methyl substitution at the α carbon of the ring. Spectra–structure relationships based on the calculations reported here are discussed, and the available experimental CD data on dissymmetric 2,5-diketopiperazine are examined in terms of our theoretical results.     

An algebraic representation of RNA secondary structures.

This paper develops mathematical methods for describing and analyzing RNA secondary structures. It was motivated by the need to develop rigorous yet efficient methods to treat transitions from one secondary structure to another, which we propose here may occur as motions of loops within RNAs having appropriate sequences. In this approach a molecular sequence is described as a vector of the appropriate length. The concept of symmetries between nucleic acid sequences is developed, and the 48 possible different types of symmetries are described. Each secondary structure possible for a particular nucleotide sequence determines a symmetric, signed permutation matrix. The collection of all possible secondary structures is comprised of all matrices of this type whose left multiplication with the sequence vector leaves that vector unchanged. A transition between two secondary structures is given by the product of the two corresponding structure matrices. This formalism provides an efficient method for describing nucleic acid sequences that allows questions relating to secondary structures and transitions to be addressed using the powerful methods of abstract algebra. In particular, it facilitates the determination of possible secondary structures, including those containing pseudoknots. Although this paper concentrates on RNA structure, this formalism also can be applied to DNA.     

An Algebraic Representation of RNA Secondary Structures

Abstract

This paper develops mathematical methods for describing and analyzing RNA secondary structures. It was motivated by the need to develop rigorous yet efficient methods to treat transitions from one secondary structure to another, which we propose here may occur as motions of loops within RNAs having appropriate sequences. In this approach a molecular sequence is described as a vector of the appropriate length. The concept of symmetries between nucleic acid sequences is developed, and the 48 possible different types of symmetries are described. Each secondary structure possible for a particular nucleotide sequence determines a symmetric, signed permutation matrix. The collection of all possible secondary structures is comprised of all matrices of this type whose left multiplication with the sequence vector leaves that vector unchanged. A transition between two secondary structures is given by the product of the two corresponding structure matrices. This formalism provides an efficient method for describing nucleic acid sequences that allows questions relating to secondary structures and transitions to be addressed using the powerful methods of abstract algebra. In particular, it facilitates the determination of possible secondary structures, including those containing pseudoknots. Although this paper concentrates on RNA structure, this formalism also can be applied to DNA     

Conformational transitions of adenylate kinase: switching by cracking

Conformational heterogeneity in proteins is known to often be the key to their function. We present a coarse grained model to explore the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins. We employ the model to study the detailed mechanism of the reversible conformational transition of Adenylate Kinase (AKE) between the open to the closed conformation, a reaction that is crucial to the protein's catalytic function. We directly observe high strain energy which appears to be correlated with localized unfolding during the functional transition. This work also demonstrates that competing native interactions from the open and closed form can account for the large conformational transitions in AKE. We further characterize the conformational transitions with a new measure Phi(Func), and demonstrate that local unfolding may be due, in part, to competing intra-protein interactions.