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R E Bruccoleri

Publications and source records attributed to R E Bruccoleri.

34 records · Page 2Linked to original sources
PubMed1990

A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity.

A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region.

Amino Acid Sequence↗
PubMed1990

Conformational sampling using high-temperature molecular dynamics.

High-temperature molecular dynamics as a method for conformational search was explored on the antigen combining site of McPC 603, a phosphorylcholine binding immunoglobulin. Simulations at temperatures of 500, 800, and 1500 K were run for 111.5, 101.7, and 76.3 ps, respectively. The effectiveness of the search was assessed using a variety of methods. For the shorter hypervariable loops, molecular dynamics explored an appreciable fraction of the conformational space as evidenced by a comparison to a simple theoretical model of the size of the conformational space. However, for the longer loops and the antigen combining site as a whole, the simulation times were too short for a complete search. The simulations at 500 and 800 K both generated conformations that minimized to energies 200 kcal/mole lower than the crystal structure. However, the 1500 K simulation produced higher energy structures, even after minimization; in addition, this highest temperature run had many cis-trans peptide isomerizations. This suggests that 1500 K is too high a temperature for unconstrained conformational sampling. Comparison of the results of high temperature molecular dynamics with a direct conformational search method, [R. E. Bruccoleri & M. Karplus (1987) Biopolymers 26, 137-168]. showed that the two methods did not overlap much in conformational space. Simple geometric measures of the conformational space indicated that the direct method covered more space than molecular dynamics at the lower temperature, but not at 1500 K. The results suggest that high-temperature molecular dynamics can aid in conformational searches.

Amino Acid Sequence↗
PubMed1990

Computer analysis of mutations that affect antibody specificity.

The mouse hybridoma cell line 40-150 secretes antibodies with high affinity toward the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40-150 A2.4, produces an antibody which carries a single residue mutation, Ser----Arg, in its heavy chain (H94) and has an altered specificity. A second-order mutant, 40-150 A2.4 P.10, produces two antibody molecules, one the same as 40-150 A2.4, the other lacking two residues at the N-terminus of its H chain, and having a specificity profile approaching that of 40-150 antibody. The N-terminus and the position H94 are distant from the antigen-binding site of the antibody; thus, the structural basis of the specificity changes was not immediately clear. Approximate structures of the 40-150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program CONGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg-H94 is substituted for Ser, Arg-H94 can hydrogen bond to side chains of Asp-H101, Arg-L46, and Asp-L55. This results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N-terminal residues increases solvent accessibility of Arg-H94. The solvation may cause a hydrogen bond between Arg-H94 and Asp-H101 to be lost, restoring the structure to one similar to that of 40-150.

Animals↗
PubMed1990

Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.

Amino Acid Sequence↗
PubMed1989

On the attribution of binding energy in antigen-antibody complexes McPC 603, D1.3, and HyHEL-5.

Using X-ray coordinates of antigen-antibody complexes McPC 603, D1.3, and HyHEL-5, we made semiquantitative estimates of Gibbs free energy changes (delta G) accompanying noncovalent complex formation of the McPC 603 Fv fragment with phosphocholine and the D1.3 or HyHEL-5 Fv fragments with hen egg white lysozyme. Our empirical delta G function, which implicitly incorporates solvent effects, has the following components: hydrophobic force, solvent-modified electrostatics, changes in side-chain conformational entropy, translational/overall rotational entropy changes, and the dilutional (cratic) entropy term. The calculated delta G ranges matched the experimentally determined delta G of McPC 603 and D1.3 complexes and overestimated it (i.e., gave a more negative value) in the case of HyHEL-5. Relative delta G contributions of selected antibody residues, calculated for HyHEL-5 complexes, agreed with those determined independently in site-directed mutagenesis experiments. Analysis of delta G attribution in all three complexes indicated that only a small number of amino acids probably contribute actively to binding energetics. These form a subset of the total antigen-antibody contact surface. In the antibodies, the bottom part of the antigen binding cavity dominated the energetics of binding whereas in lysozyme, the energetically most important residues defined small (2.5-3 nm2) "energetic" epitopes. Thus, a concept of protein antigenicity emerges that involves the active, attractive contributions mediated by the energetic antigenic epitopes and the passive surface complementarity contributed by the surrounding contact area. The D1.3 energetic epitope of lysozyme involved Gly 22, Gly 117, and Gln 121; the HyHEL-5 epitope consisted of Arg 45 and Arg 68. These are also the essential antigenic residues determined experimentally. The above positions belong to the most protruding parts of the lysozyme surface, and their backbones are not exceptionally flexible. Least-squares analysis of six different antibody binding regions indicated that the geometry of the VH-VL interface beta-barrel is well conserved, giving no indication of significant changes in domain-domain contacts upon complex formation.

Amino Acids↗
PubMed1989

On the molecular nature of "restrictive" antigenic elements present on major histocompatibility complex (MHC) proteins.

By analogy with the way in which antibodies recognize their specific antigens, it appears likely that T-cell receptors recognize peptides presented by MHC molecules as composite epitopes involving the presented peptide and portions of the MHC molecules. We extend here the analogy to attempt to define which portions of the MHC molecules are most accessible to the TcR and thereby most likely to participate in the binding. We suggest that the alpha chain segments 56-60, 73-77, 149-153 and 158-162, which are most protruding according to accessibility calculations, are likely candidates for the interaction. Again, by analogy with antibodies, we further propose that TcR recognition may involve: (1) an "active" recognition process, focussed on a few residues which provide the major contribution to the binding energy, and (2) a "passive" recognition process, in which the relatively large contact areas between TcR and the composite epitope primarily need to be compatible with one another rather than to contribute significantly to binding energy.

Binding Sites↗
PubMed1988

Structure of antibody hypervariable loops reproduced by a conformational search algorithm.

The antigen-combining site of antibody molecules consists of six separate loops supported by a conserved beta-sheet framework; antibody specificity arises from length and sequence variation of these 'hypervariable' loops and can be manipulated by transferring sets of loops between different frameworks. Irregular loops are the most difficult parts of protein structure to understand and to model correctly. Here, we describe two computer experiments where all the hypervariable loops were deleted from X-ray structures of mouse immunoglobulins and reconstructed using the conformational search program CONGEN. A protocol was developed for reconstruction of the hypervariable loops in McPC 603 antibody. Calculated loop conformations were generated and a model of the combining site was built from selected low-energy conformations. We then modelled hypervariable loops in another antibody molecule, HyHEL-5. Both models agreed well with the known crystal structures. Our results hold out promise for the success of future modelling studies of complete antigen-combining sites from amino acid sequences.

Algorithms↗
PubMed1988

Criteria that discriminate between native proteins and incorrectly folded models.

Various theoretical concepts, such as free energy potentials, electrostatic interaction potentials, atomic packing, solvent-exposed surface, and surface charge distribution, were tested for their ability to discriminate between native proteins and misfolded protein models. Misfolded models were constructed by introducing incorrect side chains onto polypeptide backbones: side chains of the alpha-helical hemerythrin were modeled on the beta-sheeted backbone of immunoglobulin VL domain, whereas those of the VL domain were similarly modeled on the hemerythrin backbone. CONGEN, a conformational space sampling program, was used to construct the side chains, in contrast to the previous work, where incorrect side chains were modeled in all trans conformations. Capability of the conformational search procedure to reproduce native conformations was gauged first by rebuilding (the correct) side chains in hemerythrin and the VL domain: constructs with r.m.s. differences from the x-ray side chains 2.2-2.4 A were produced, and many calculated conformations matched the native ones quite well. Incorrectly folded models were then constructed by the same conformational protocol applied to incorrect amino acid sequences. All CONGEN constructs, both correctly and incorrectly folded, were characterized by exceptionally small molecular surfaces and low potential energies. Surface charge density, atomic packing, and Coulomb formula-based electrostatic interactions of the misfolded structures and the correctly folded proteins were similar, and therefore of little interest for diagnosing incorrect folds. The following criteria clearly favored the native structures over the misfolded ones: 1) solvent-exposed side-chain nonpolar surface, 2) number of buried ionizable groups, and 3) empirical free energy functions that incorporate solvent effects.

Computer Simulation↗
PubMed1988

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

Base Sequence↗
PubMed1988

An improved algorithm for nucleic acid secondary structure display.

An improved algorithm for the display of nucleic acid secondary structures is presented. It is particularly suitable for large sequence segments and it automatically generates an aesthetically pleasing display of the structure with very limited overlap of strands. Structural similarities in different structures are conserved in the display thus greatly aiding structural homology comparisons. Using the algorithm, we illustrate the effect of ribosome translocation on the secondary structure of a rat neuropeptide messenger RNA.

Algorithms↗
PubMed1987

Correlation among sites of limited proteolysis, enzyme accessibility and segmental mobility.

The relationship among accessibility to an enzyme, flexibility, and limited proteolysis was explored. Regions accessible to large probes, comparable in size to proteolytic enzymes, were computed in the crystallographic structures of thermolysin, trypsinogen and ribonuclease. Positions of these accessible regions were compared with sites of autolytic/proteolytic attacks, and with locations of flexible backbone segments. All the proteolytic sites were found to be exceptionally accessible. Most of them were also flexible, but at least one prominent site in trypsinogen appeared to be rigid. Thus, surface exposure seems to be more essential to proteolysis than flexibility.

Biological Availability↗
PubMed1986

Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains).

We evaluated surface areas on proteins that would be accessible to contacts with large (1-nm radius) spherical probes. Such spheres are comparable in size to antibody domains that contain antigen-combining sites. We found that all the reported antigenic sites correspond to segments particularly accessible to a large sphere. The antigenic sites were also evident as the most prominently exposed regions (hills and ridges) in contour maps of the solvent-accessible (small-probe) surface. In myoglobin and cytochrome c, virtually all of the van der Waals surface is accessible to the large probe and therefore potentially antigenic; in myohemerythrin, distinct large-probe-inaccessible, and nonantigenic, surface regions are apparent. The correlation between large-sphere-accessibility and antigenicity in myoglobin, lysozyme, and cytochrome c appears to be better than that reported to exist between antigenicity and segmental flexibility; that is, surface regions that are rigid often constitute antigenic epitopes, whereas some of the flexible parts of the molecules do not appear antigenic. We propose that the primary reason why certain polypeptide-chain segments are antigenic is their exceptional surface exposure, making them readily available for contacts with antigen-combining sites. Exposure of these segments frequently results in high mobility and, in consequence, to the reported correlation between antigenicity and segmental flexibility.

Cytochrome c Group↗
PubMed1984

Twisted hyperboloid (Strophoid) as a model of beta-barrels in proteins.

Using a least-squares fitting procedure, polypeptide backbones of one parallel and seven antiparallel beta-barrels were approximated with various curved surfaces. Although the hyperboloid gave better approximations to all the beta-barrel backbones than the ellipsoid, elliptical cylinder or catenoid, the best approximations were obtained with a novel surface, a twisted hyperboloid (strophoid). The root-mean-square errors between individual beta-barrels and the fitted strophoid surfaces ranged from 0.75 A to 1.64 A. The parameters which determine the strophoid surface allow groups of beta-barrel shapes to be defined according to their barrel twists (i.e. angles subtended by directions of the long axis of cross-section at the top and the bottom of the barrel), course of elliptical cross-sections (either monotonically increasing along the barrel axis, as in cones, or having a middle "waist", as in hyperboloids), and types of backbone curvatures (either convex or concave). The curvatures at individual points of strophoid surface are local, variable quantities related to the local helicity (coil) of the polypeptide backbone, in contrast to values of beta-sheet twist (i.e. dihedral angles subtended by adjacent beta-strands) known to be virtually identical in all the beta-sheets. The variability found in parameters such as barrel shapes and curvatures suggests that simple models (isotropically stressed surfaces, principle of minimal surface tension) proposed in the past to account for beta-barrel shapes are not sufficient. Rather, the complex nature of best-fit theoretical surfaces points to an important role played by a local variability of the forces involved.

Azurin↗