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PubMed2002

Global and local motions in ribonuclease A: a molecular dynamics study.

The understanding of protein dynamics is one of the major goals of structural biology. A direct link between protein dynamics and function has been provided by x-ray studies performed on ribonuclease A (RNase A) (B. F. Rasmussen et al., Nature, 1992, Vol. 357, pp. 423-424; L. Vitagliano et al., Proteins: Structure, Function, and Genetics, 2002, Vol. 46, pp. 97-104). Here we report a 3 ns molecular dynamics simulation of RNase A in water aimed at characterizing the dynamical behavior of the enzyme. The analysis of local and global motions provides interesting insight on the dynamics/function relationship of RNase A. In agreement with previous crystallographic reports, the present study confirms that the RNase A active site is constituted by rigid (His12, Asn44, Thr45) and flexible (Lys41, Asp83, His119, Asp121) residues. The analysis of the global motions, performed using essential dynamics, shows that the two beta-sheet regions of RNase A move coherently in opposite directions, thus modifying solvent accessibility of the active site, and that the mixed alpha/3(10)-helix (residues 50-60) behaves as a mechanical hinge during the breathing motion of the protein. These data demonstrate that this motion, essential for RNase A substrate binding and release, is an intrinsic dynamical property of the ligand-free enzyme.

Animals↗

PubMed1989

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies.

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.

Magnetic Resonance Spectroscopy↗

PubMedDate not supplied

A molecular dynamics investigation of the elastomeric restoring force in elastin.

A repetitive polypentapeptide organized as a connected chain of beta-bends is believed to be an important structural element of elastin, the major elastomer in biological systems. Molecular dynamics simulations were carried out on hydrated polymers of (Val-Pro-Gly-Val- Gly)18 at various extensions. Analysis of the fluctuations of backbone angles in relaxed elastin showed that particularly large-amplitude torsional motions occur in phi and psi angles of residues connecting sequentially adjacent hairpin bends. Many such motions reflect peptide plane librations that result from anticorrelated crankshaft rotations of psi i and phi i+1. These effects were much reduced in stretched polymer models. The conformational entropy of relaxed and stretched elastin models was estimated using a treatment due to Meirovitch, and gave a calculated decrease in entropy of about 1 cal/mol deg when the polymer was stretched to 1.75 times its original length. There are large changes in solvent-accessible surface area during the initial stages of elastin stretching. Collectively these results suggest that hydrophobic interactions make contributions to elastin entropy at low extensions, but that librational mechanisms make larger contributions to the elastic restoring force at longer extensions.

Amino Acid Sequence↗

PubMed1991

Premelting thermal fluctuational base pair opening probability of poly(dA).poly(dT) as predicted by the modified self-consistent phonon theory.

We employ a mean field, modified, self-consistent phonon theory to evaluate the single base-pair opening rate and the probability of a base pair in the amino proton exchangeable state for the homopolymer poly(dA).poly(dT) at temperatures below the helix-coil transition region. Our calculated premelting single base-pair opening probabilities are in general agreement with several available experimental estimates from imino proton exchange and formaldehyde-induced DNA melting measurements. These calculated opening probabilities, however, are in disagreement with the prediction of the helix-coil transition theory. Possible reasons for the differences are discussed, especially the possible different definition of a meaningful open state in the premelting region. The premelting open state of the modified self-consistent phonon approximation theory seems to be appropriate to describe a solvent-accessible open configuration that is sufficient to facilitate important chemical reactions such as imino proton exchange and formaldehyde reaction with the bases. This can be compared with the completely unstacked open state of the helix-coil transition theory originally defined in the helix-coil transition region. We propose that the amino proton exchangeable state is different from the open state associated with melting and only involves the breaking of the amino interbase H bond. The agreement between the calculated and experimentally estimated probability of a base pair in the amino proton exchangeable state seems to support this hypothesis.

Base Composition↗

PubMed1993

Necessary conditions for avoiding incorrect polypeptide folds in conformational search by energy minimization.

Low energy conformations have been generated for melittin, pancreatic polypeptide, and ribonuclease S-peptide, both in the vicinity of x-ray structures by energy refinement and by an unconstrained search over the entire conformational space. Since the structural polymorphism of these medium-sized peptides in crystal and solution is moderate, comparing the calculated conformation to x-ray and nmr data provides information on local and global behavior of potential functions. Local analysis includes standardization calculations, which show that models with standard geometry can approximate good resolution x-ray data with less than 0.5 A rms deviation (RMSD). However, the atomic coordinates are shifted up to 2 A RMSD by local energy minimization, and thus 2 A is generally the smallest RMSD value one can target in a conformational search using the same energy evaluation models. The unconstrained search was performed by a buildup-type method based on dynamic programming. To accelerate the generation of structures in the conformational search, we used the ECEPP potential, defined in terms of standard polypeptide geometry. A number of low energy conformations were further refined by relaxing the assumption of standard bond lengths and bond angles through the use of the CHARMM potential, and the hydrophobic folding energies of Eisenberg and McLachlan were calculated. Each conformation is described in terms of the RMSD from the native, hydrogen-bonding structure, solvent-accessible surface area, and the ratio of surfaces corresponding to nonpolar and polar residues. The unconstrained search finds conformations that are different from the native, sometimes substantially, and in addition, have lower conformational energies than the native. The origin of deviations is different for each of the three peptides, but in all examples the refined x-ray structures have lower energies than the calculated incorrect folds when (1) the assumption of standard bond lengths and bond angles is relaxed; (2) a small and constant effective dielectric permittivity (epsilon < 10) is used; and (3) the hydrophobic folding energy is incorporated into the potential.

Amino Acid Sequence↗

PubMed1993

Conformational preference functions for predicting helices in membrane proteins.

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the alpha-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in alpha-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 alpha-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known alpha-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements.

Animals↗

PubMed1993

Molecular dynamics of sickle and normal hemoglobins.

Molecular dynamics (MD) simulations have been carried out for 62.5 ps on crystal structures of deoxy sickle cell hemoglobin (HbS) and normal deoxy hemoglobin (HbA) using the CHARMM MD algorithm, with a time step of 0.001 ps. In the trajectory analysis of the 12.5-62.5 (50 ps) simulation, oscillations of the radius of gyration and solvent-accessible surface area were calculated. HbS exhibited a general contraction during the simulation, while HbA exhibited a nearly constant size. The average deviations of simulated structures from the starting structures were found to be 1.8 A for HbA and 2.3 A for HbS. The average rms amplitudes of atomic motions (atomic flexibility) were about 0.7 A HbA and about 1.0 A for HbS. The amplitudes of backbone motion correlate well with temperature factors derived from x-ray crystallography. A comparison of flexibility between the alpha- and beta-chains in both HbA and HbS indicates that the beta-chains generally exhibited greater flexibility than the alpha-chains, and that the HbS beta-chains exhibit greater flexibility in the N-terminal and D- and F-helix regions than do those of HbA. The average amplitude of backbone torsional oscillations was about 9 degrees, a value comparable with that of other simulations, with enhanced torsional oscillation occurring primarily at the ends of helices or in loop regions between helices. Comparison of atomic flexibility and torsional oscillation results suggests that the increased beta-chain flexibility results from relatively concerted motions of secondary structure elements. The increased flexibility may play an important role in HbS polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Simulation↗

PubMed1993

Synthesis and conformational study of peptides possessing helically arranged delta ZPhe side chains.

Z-Dehydrophenylalanine (delta ZPhe) possessing four oligopeptides, Boc-(L-Ala-delta ZPhe-Aib)n-OCH3 (n = 1-4: Boc, t-butoxycarbonyl; Aib, alpha-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by 1H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. 1H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L-Ala-delta ZPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2-4 have a 4-->1 hydrogen-bonding pattern characteristic of 3(10)-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the delta ZPhe chromophores. The corresponding CD spectra of the peptides n = 2-4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2-4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3(10)-helix that contains three residues per turn, and that their side chains of delta ZPhe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be approximately 5.5 A. The chemical shifts of the delta ZPhe side-chain protons (H beta and aromatic H) for the peptides n = 2-4 indicated anisotropic shielding effect of neighboring phenyl group (s); the effect also supports a regular arrangement of the delta ZPhe side chains along the helical axis.

Amino Acid Sequence↗

PubMed1994

Modified chemotactic peptides: synthesis, conformation, and biological activity of For-Thp-Leu-delta ZPhe-OMe.

For-Thp-Leu-delta ZPhe-OMe (2), an analogue of the chemotactic tripeptide For-Met-Leu-Phe-OMe, containing 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and (Z)-2,3-didehydrophenylalanine (delta ZPhe) as achiral, conformationally restricted mimics of Met and Phe, respectively, has been synthesized. In the crystal the new formyltripeptide adopts a type I beta-turn conformation stabilized by a weak H bond between the formylic oxygen and the delta ZPhe NH. 1H-nmr analysis based on NH solvent accessibility and nuclear Overhauser effect experiments suggests that the beta-turn is not preferred in CDCl3 solution where a gamma-turn, centered at the Thp residue, prevails. The biological activity of 2 has been determined on human neutrophils and compared to that of previously studied analogues. The tripeptide 2 is practically unable to elicit superoxide anion production and lysozyme release, while slight, but not statistically significant activity was induced in chemotaxis. The role of the orientation of the aromatic ring with respect to the backbone adjacent atoms is discussed.

Amino Acid Sequence↗

PubMed1995

Slow conformational exchange in DNA minihairpin loops: a conformational study of the circular dumbbell d .

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson-Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decamer 5'-d -3' (I) is studied in solution by means of nmr spectroscopy. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson-Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5'-GTTC-3' loop with only one remaining solvent-accessible hydrogen bond between NH alpha of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5'-CTTG-3' loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5'-3' loop-stem junction in the 5'-GTTC-3' loop. The two conformers, L2L2 and L2L4, occur in slow equilibrium (2-20 s-1). Moderate line broadening of specific 1H, 13C, and 31P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T1 relaxation measurements, chemical-shift considerations, and complete band-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational states in the anti-syn equilibrium.

Base Sequence↗

PubMed2006

Investigation of protein retention and selectivity in HIC systems using quantitative structure retention relationship models.

In the present work, the effect of stationary phase resin chemistry and protein physicochemical properties on protein binding affinity in hydrophobic interaction chromatography (HIC) was investigated using linear gradient chromatography and quantitative structure-retention relationship (QSRR) modeling. Linear gradient experiments were carried out for a set of model proteins on four different HIC resins having different backbone and ligand chemistry. The retention data exhibited significant differences in protein binding affinity, not only across the phenyl and butyl ligand chemistries, but also for the different backbone chemistries found in the Sepharose (cross-linked agarose) and the Toyopearl 650 M (polymethacrylate) series of resins. QSRR models based on a Support Vector Machine (SVM) approach were developed for the linear retention data using molecular descriptors based on protein crystal structure and primary sequence information as well as a set of new hydrophobicity descriptors based on the solvent accessible protein surface area. The results indicate that the QSRR models were successfully able to capture and selectivity predict the changes observed in these systems. Furthermore, the new descriptors resulted in physically interpretable models of protein retention and provided insights into the factors influencing protein affinity in these different HIC systems. The approach put forth in this study provides a framework for developing predictive tools and for gaining insight into protein selectivity in hydrophobic interaction chromatography.

Chromatography, Gel↗

PubMed2003

A new strategy for the design of water-soluble synthetic receptors: specific recognition of DNA intercalators and diamines.

Water-soluble zinc bisporphyrin receptors 1 and 2 having two Lewis acidic sites (zinc) in the hydrophobic environment consisting of alkyl chains and a bisporphyrin framework, and covered with hydrophilic exterior (twelve or eighteen carboxyl groups) were prepared. The receptors show high affinity for diamines and DNA intercalators in water where the binding constants K(a) are of the order of 10(7) and 10(8) M(-1), respectively. Diamines and DNA intercalators are bound to the receptor through different mechanisms. Diamines are bound through hydrophobic interactions and zinc-nitrogen interactions, while DNA intercalators are bound through hydrophobic interactions and charge-transfer interactions. Flexible alkyl chains can make van der Waals contact with guests and create a hydrophobic environment around the bound guest by an induced-fit-type mechanism. For the binding of DNA intercalators, the following features are noteworthy: 1). Binding constants are similar between the zinc porphyrins and zinc-free porphyrins; 2). the binding constant is larger for the guest having the lower LUMO; this indicates the important contribution of charge-transfer interactions to binding; 3). the hydrophobic and cationic nature of DNA intercalators is substantially important, and 4). higher ionic strength reduced the binding affinities; this shows a moderate contribution of electrostatic interactions. The conformational instability of the receptors also contributes to the tight binding: hydrophobic and electrostatic interactions cannot both be favorable at the same time in the guest-free receptor. Enthalpy-entropy compensation observed for the binding of diamines and DNA intercalators is characterized by a relatively small slope (alpha=0.74) and a large intercept (beta=7.75 kcal mol(-1)) in the DeltaH degrees versus TDeltaS degrees plot; this shows that a conformational change of receptors and a significant desolvation occur upon binding. The receptor can competitively bind to propidium iodide to deprive DNA of the intercalated propidium iodide. These features of water-soluble receptors consisting of a rigid framework and flexible side chains with a large solvent-accessible area are in contrast to highly preorganized rigid receptors, and they can provide useful guidelines for rational design of induced-fit artificial receptors in water.

Binding, Competitive↗

PubMed1997

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

ADP Ribose Transferases↗

PubMed1986

Location of 'continuous' antigenic determinants in the protruding regions of proteins.

A simple method is described to locate 'antigenic' peptides from the alpha-carbon co-ordinates of a protein, based on protrusion from the protein's globular surface. A good correlation is found between those parts of a protein which protrude and the experimentally determined antigenic peptides in myoglobin, lysozyme and myohemerythrin. A comparison is made between the use of protrusion index, mobility, solvent accessibility and hydrophilicity for predicting the most likely antigenic peptides.

Epitopes↗

PubMed1989

Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha-helix and beta-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Protein-protein interactions directing resolvase site-specific recombination: a structure-function analysis.

Recombination catalyzed by the gamma delta resolvase requires assembly of a nucleo-protein complex, the synaptosome, whose structure is determined by resolvase-res and resolvase-resolvase interactions. In crystals of the resolvase catalytic domain, monomers of resolvase were closely associated with one another across three different dyad axes; one of these subunit contacts was shown to be an essential inter-dimer interaction. To investigate the relevance of the remaining two interfaces, we have made site-directed mutations at positions suggested by the structure. Cysteine substitutions were designed to link the interfaces covalently, mutations to arginine were used to disrupt intersubunit contacts, and mutations to tryptophan were used to study the hydrophobicity and solvent accessibility of potential interfaces by fluorescence quenching. Characterization of the mutant proteins has allowed us to identify the dimer interface of resolvase and to assign a structural role to a second intersubunit contact. The data presented here, together with our previous results, suggest that all three of the dyad-related intersubunit interactions observed in the crystal play specific roles in synapsis and recombination.

Arginine↗

PubMed1993

Effect of the conformation and orientation of adsorbed fibronectin on endothelial cell spreading and the strength of adhesion.

The effect of surface hydrophobicity upon the conformation of the cell binding domain of fibronectin (Fn) and the influence of Fn conformation on bovine aortic endothelial cell (BAEC) adhesion were examined. The free sulfhydryl group of Fn located near the cell binding domain was selectively labeled with acrylodan, a polarity sensitive fluor. Fluorescence emission was monitored in solution and upon adsorption to hydrophilic glass and hydrophobic silanized glass. The acrylodan-labeled Fn emission maximum shifted to longer wavelengths upon adsorption and the shift was greater for acrylodan-labeled Fn adsorbed to hydrophilic glass than hydrophobic silane, suggesting that the acrylodan was in a more solvent accessible environment on glass than silane. BAEC, suspended in serum-free medium, attached for 15 or 120 min onto glass or silane surfaces containing preadsorbed Fn, after which cell spreading and the strength of adhesion in a parallel plate flow chamber were measured. Cell spreading was similar on both surfaces after 15 min attachment, but BAECs were more spread on glass than silane after 120 min. At low surface concentrations of Fn, BAECs were more adherent on glass than silane. At higher surfaces concentrations, adhesion was similar. After a 2-h incubation in serum-free medium, cells on glass showed more extensive development of focal contacts as determined by immunofluorescent staining for vinculin. Cell adhesion under flow was reduced on silane by inhibition of protein synthesis with cycloheximide, suggesting that cell attachment to silane was promoted by cellular synthesis of Fn. The results indicate that changes in the conformation of the Fn cell binding domain affect Fn affinity for its cell surface receptor.

Adsorption↗

PubMed2001

Three-dimensional structural analysis of fibronectin heparin-binding domain mutations.

Using recombinant fibronectin proteins containing the V region and two point mutations in the high-affinity heparin-binding domain, we previously showed that these domains modulate tumor cell invasion as well as proteinase expression and apoptosis in human fibroblasts. Structurally, the wildtype counterparts to these two point mutations, together with four other discontinuous, positively charged residues, form a cationic cradle in domain III-13 of fibronectin that binds heparin. We constructed a three-dimensional model of this cationic cradle and determined whether the two engineered point mutations in the heparin-binding domain would alter this cradle conformation, thus explaining the altered cell behavior. Our model of fibronectin domain III-13 was generated from a template of the three-dimensional structure of a homologous (25% identity) domain, III-3, from tenascin. The amino acid sequences of III-13 that differed from tenascin III-3 were replaced, and side chains for positively charged arginines 6 and 7 were substituted with uncharged threonines. The model revealed that the two mutated threonine residues were solvent accessible, readily accommodated as part of an antiparallel beta strand, and remained part of the three-dimensional cradle. These models suggest that the two point mutations in the heparin-binding domain of fibronectin III-13 alter cell function probably through changes in charge and not through changes in the conformational structure of the cationic cradle. J. Cell. Biochem. Suppl. 36: 156-161, 2001.

Amino Acid Sequence↗