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Biomedical subjects

A C Martin

Publications and source records attributed to A C Martin.

At least 37 records · Page 2Linked to original sources

Protein folds and functions.

BACKGROUND: The recent rapid increase in the number of available three-dimensional protein structures has further highlighted the necessity to understand the relationship between biological function and structure. Using structural classification schemes such as SCOP, CATH and DALI, it is now possible to explore global relationships between protein fold and function, something which was previously impractical. RESULTS: Using a relational database of CATH data we have generated fold distributions for arbitrary selections of proteins automatically. These distributions have been examined in the light of protein function and bound ligand. Different enzyme classes are not clearly reflected in distributions of protein class and architecture, whereas the type of bound ligand has a much more dramatic effect. CONCLUSIONS: The availability of structural classification data has enabled this novel overview analysis. We conclude that function at the top level of the EC number enzyme classification is not related to fold, as only a very few specific residues are actually responsible for enzyme activity. Conversely, the fold is much more closely related to ligand type.

Binding Sites↗

Genetic, structural and functional properties of an IgG DNA-binding monoclonal antibody from a lupus patient with nephritis.

Antibodies binding to double-stranded (ds) DNA are strongly associated with renal involvement in patients with systemic lupus erythematosus (SLE). We have generated two new IgG DNA-binding monoclonal antibodies (mAb), RH-14 and DIL-6, from the peripheral blood lymphocytes of two SLE patients with glomerulonephritis using the heteromyeloma cell line CB-F7. RH-14 is an IgG1 lambda antibody which also bound to single-stranded DNA, histones and nucleosomes. DIL-6 is an IgG3 lambda antibody with restricted antigen binding specificity. cDNA encoding the variable regions of the heavy (V(H)) and light (V(L)) chains of RH-14 was sequenced and the antigen binding site of this mAb was computer modelled. Sequence analysis of V(H) and V(L) regions of RH-14 showed that V(H) is derived from germ-line gene V3-7, a member of the V(H)3 family, and V(L) is derived from DPL 11, a member of the V(lambda)2 family. Somatic mutations and basic amino acid residues are identified in the complementarity-determining regions of both V(H) and V(L) regions. The nephritogenic properties of these mAb were analyzed by implanting and growing the hybridoma cells secreting the mAb in the peritoneum of SCID mice. The animals that received the RH-14 hybridoma produced higher levels of proteinuria (3 to > or = 4) (p < 0.001) compared to the groups that received DIL-6 (trace to > or = 1) or CB-F7 (trace). Electron microscopy of kidney sections from all the RH-14-implanted animals showed granular immunoglobulin deposition in the renal glomerular capillaries and mesangium. In this study we have shown for the first time using electron microscopy that a human IgG anti-dsDNA mAb, RH-14, is nephritogenic and that deposition of such an antibody alone is sufficient to induce renal damage.

Amino Acid Sequence↗

Dihydrofolate reductase: a potential drug target in trypanosomes and leishmania.

Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.

Amino Acid Sequence↗

Assessment of comparative modeling in CASP2.

An assessment is presented for all submissions to the comparative modeling challenge in the 1996 Critical Assessment of Structure Prediction (CASP2). Of the original 12 target structures, 9 were solved prior to the meeting: 8 by X-ray crystallography and 1 by NMR spectroscopy. These targets varied over a large range of difficulty, as assessed by the percentage sequence identity with the principal parent structure, which ranged from 20% up to 85%. The overall quality of the models reflected the similarity of the principal parent. As expected, when the sequence alignment was correct, the core was accurately modeled, with the largest deviations occurring in the loops. Models were built which gave C alpha root-mean-square deviations (RMSDs) compared with the observed structure of < 1 A for targets with high parental similarity; even at 26% sequence identity, the best model structures had C alpha deviations of only 2.2 A. Overall, these deviations are comparable with those observed between the parent structure and the target, but locally there are several examples where the model approaches closer to the target than does the parent. There were three targets below 25% sequence identity, and the models generated for these targets were, in general, significantly less accurate. This principally reflects errors in the alignment which, if systematically shifted, can generate C alpha RMSDs > 18 A. Compared with CASP1, the geometry of the models was significantly improved with no D-amino acids. By far the major contribution to RMSD error was the alignment accuracy, which varied from 100% down to 7% over the range of targets. In the structurally variable regions, global shifts, caused by hinge bending, were the major source of error, giving significantly lower local RMSDs than global RMSDs. In over 50% of these noncore regions, the difference between global and local RMSDs was more than 3 A, and was as high as 10 A for one structurally variable region. For the side chains, the chi 1 RMSDs are strongly correlated with the C alpha RMSDs. For models with C alpha deviations less than 1 A, on average 78.5% of side chains are placed in the correct rotamer, although the chi 1 RMSDs, though clearly better than random, were disappointing at around 46 degrees. As the backbone deviations increased, the side chain placement became less accurate, with an average chi 1 RMSD of 75 degrees on a 1.5-2.5 A C alpha backbone (average 51.4% correct rotamer). Refinement by energy minimization or molecular dynamics made only minor adjustments to improve local geometry and generally made small, but not significant, improvements to the RMSD. In total, 19 groups submitted 62 models (89 coordinate sets) that could be assessed. Most modelers used manual adjustments to sequence alignments and, in general, good alignments were obtained down to 25% sequence identity. The modeling methods ranged from "classical" modeling, involving core building followed by loop and side chain addition, to more sophisticated approaches based on probability distributions, Monte Carlo sampling or distance geometry. For each target, several groups produced equally good models, given the expected errors in the structures (about 0.5 A). No one method came out as clearly superior, although the approaches that inherit directly from the parents generally performed better than the more radical techniques. However, for each target there were some poor models, usually reflecting a poor sequence alignment, and the range of accuracy for each target is therefore large. Fully automated methods are able to perform very well for "easy" targets (85% sequence identity with parent), but when modeling using a distantly related parent, care and expertise, especially in performing the alignment, still appear to be important factors in generating accurate models.

Models, Molecular↗

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

Amino Acid Sequence↗

Structural families in loops of homologous proteins: automatic classification, modelling and application to antibodies.

Loop regions of polypeptide in homologous proteins may be classified into structural families. A method is described by which this classification may be performed automatically and "key residue" templates, which may be responsible for the loop adopting a given conformation, are defined. The technique has been applied to the hypervariable loops of antibodies and the results are compared with the previous definition of canonical classes. We have extended these definitions and provide complete sets of structurally determining residues (SDRs) for the observed clusters including the first set of key residues for seven-residue CDR-H3 loops.

Antibodies↗

Antibody-antigen interactions: contact analysis and binding site topography.

We have analysed antigen-contacting residues and combining site shape in the antibody Fv and Fab crystal structures now available from the Protein Data Bank. Antigen-contacting propensities are presented for each antibody residue, allowing a new definition for the complementarity determining regions (CDRs) to be proposed based on observed antigen contacts. Contacts are more common at CDR residues which are located centrally within the combining site; some less central CDR residues are only contacted by large antigens. Non-contacting residues within the CDRs coincide with residues identified by Chothia and co-workers as important in defining "canonical" conformations. An objective means of classifying protein surfaces by gross topography has been developed and applied to the antibody combining site surfaces. The surfaces have been clustered into four topographic classes: concave and moderately concave (mostly hapten binders), ridged (mostly peptide binders) and planar (mostly protein binders). We have determined the topographic classes for ten pairs of complexed and uncomplexed antibody-antigen crystal structures; four change topographic class on complexation. The results will be of use in antibody engineering, antigen docking and in clinical immunology. To demonstrate one application, we show how the data can be used to locate the antigen binding pocket on antibody structures.

Antibodies↗

Accessing the Kabat antibody sequence database by computer.

The Kabat antibody sequence database has for many years been the primary site for depositing sequence information on antibodies and other proteins of immunological interest. The chief drawback of this database has been that it has only been available in the form of a printed book (Kabat et al., Sequences of Proteins of Immunological Interest, 1991). These data have recently become available on the global computer Internet, but no method of searching the data has, as yet, been provided. Here, the development of a specialized database program for accessing the antibody data is described. This database software has been made accessible over the World Wide Web, together with a program which allows a novel antibody sequence to be tested against the Kabat sequence database, to identify unusual features of an antibody sequence which may represent cloning artifacts or sequencing errors.

Amino Acid Sequence↗

Long loops in proteins.

In proteins, loop regions which connect secondary structures are generally short (80% are <10 amino acids long). However, long loops, defined here as > or = 10 residues, do occur. Two types of long loop may be distinguished: those which connect adjacent regions of secondary structure ('long-closed') and those which connect distant secondary structures ('long-open'). Only 5% of all loops in proteins fall into the long-open class. Inspection of these unusual long-open loops reveals a higher percentage of proline residues and more helix-helix linkages than expected. Exposure to solvent is similar for long-open and long-closed loops. The role of these loops in protein folding is discussed.

Amino Acid Sequence↗

Prediction of hypervariable CDR-H3 loop structures in antibodies.

The structure of the most variable antibody hypervariable loop, CDR-H3, has been predicted from amino acid sequence alone. In contrast to other approaches predictions are made for loop lengths up to 17 residues. The predictions have been achieved using artificial neural networks which are trained on a large set of loops from the Brookhaven Protein Databank which have structures similar to CDR-H3. The loop structures are described by the two backbone dihedral angles phi and psi for each residue. For 21 CDR-H3 loops unique to the neural network, the prediction of dihedral angles leads to an average root mean square deviation in the Cartesian coordinates of 2.65 A. The present method, when combined with existing modelling protocols, provides an important addition to the structural prediction of the complementarity determining regions of antibodies.

Amino Acid Sequence↗

Breastfeeding: perceived barriers and benefits/enhancers in a rural and urban setting.

This study investigated differences in perceived benefits/enhancers and barriers between women who planned to breastfeed, those who were uncertain, and those who did not plan to breastfeed. The volunteers were obtained from rural (60) and urban (82) public health department prenatal clinics. Fifty-three percent of the women were married, the average age was 23 years, 25% had not graduated from high school, 38% were black, and 46% were planning to return to work. The breastfeeding inventory consisted of 13 items to measure benefits and enhancers, 15 to measure barriers, and an open-ended question. Cronbach's alphas for internal consistency for benefits and enhancers and barriers were 0.88 and 0.83 respectively. Analysis of variance found significant differences among the three groups on perceived benefits and barriers. Rural and urban differences existed in perceived benefits/enhancers, but not in perceived barriers. In a multiple discriminant function analysis that included demographic characteristics, the most important predictor of breastfeeding decision was perceived benefits.

Adolescent↗

MoG: molecular graphics software for the Commodore Amiga.

As recently described by Garavelli, the Commodore Amiga 3000 computer is "nearly ideal" for desktop molecular modeling. The chief drawback to date, has been the lack of suitable software. This paper describes a new desktop molecular modeling package, MoG, which is suitable for both research and educational use. The speed of the Amiga 3000 means that MoG competes very favorably with software on IBM-PC machines, and its graphics capabilities allow excellent space-filling representations. The availability of cheap software-compatible home-computer versions of the Amiga places interactive molecular graphics within the reach of many senior high-school students, undergraduates and graduate students.

Antibodies↗

The prediction and characterization of metal binding sites in proteins.

The rational engineering of novel functions into proteins can only be attempted when the underlying structural scaffold on which the new function is displayed and the structure of the target protein are both well understood. To introduce functions mediated by metals it is therefore necessary to identify the principal liganding residues for the chosen metal, the required architecture of the metal-ligand complex and sites within the target protein that could accommodate such sites. Here we present a method that applies structural information from the protein data bank to the ab initio design and characterization of novel metal binding sites. The prediction method has been tested on 28 metalloprotein structures from the Brookhaven Protein Data Bank. It successfully identified > 90% of the metal binding sites. In addition, we have used the method to design and characterize zinc binding sites in two antibody structures. Metal binding studies on one of these putative metalloantibodies showed metal binding, confirming the predictive power of the method.

Algorithms↗

Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody.

A three-dimensional (3D) molecular model of the antigen-combining site of a bovine anti-testosterone monoclonal antibody has been constructed. In the model, the CDRs, and a single heavy chain framework region residue (Trp47), associate to form a hydrophobic cavity large enough to accommodate a single molecule of testosterone. Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxyl group exposed to solvent. Using the model and data from binding studies, we predicted that the cavity forms the antibody's paratope and on binding testosterone a hydrogen bond is formed between Tyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone. This prediction has subsequently been tested by site-directed mutagenesis. An antibody with phenylalanine in place of tyrosine at position 97 in CDR-H3 has its affinity reduced by approximately 800 fold. The reduction in binding energy associated with the reduced affinity has been calculated to be 3.9 kcal/mol which is within the range (0.5-4.0 kcal/mol) expected for the loss of a single hydrogen bond. The model has been used to suggest ways of increasing the antibody's affinity for testosterone.

Amino Acid Sequence↗

Developing national guidelines for nurse practitioner education: an overview of the product and the process.

In 1988, the National Organization of Nurse Practitioner Faculties appointed an ad hoc education committee (AHEC) to review existing nurse practitioner (NP) education curricula and to formulate national guidelines. An overview of the new NP guidelines are presented along with the philosophy, content, objectives, and competencies that are viewed as the foundation for NP education today. The guidelines are presented within the structure outlined in Brykczynski's (1985) research about NP practice. Thus, as a "product" of the Advanced Nursing Practice: Nurse Practitioner Curriculum Guidelines (NPCG), a new NP graduate for the 1990s could be described as one who demonstrates beginning competencies in primary health care theory and research within the five domains of practice identified by Brykczynski.

Clinical Competence↗

Molecular modeling of antibody combining sites.

Each of the six CDRs of Gloop2 is shown with the modeled structure in. Overall, the results obtained using the combined algorithm are similar in accuracy to those achieved using the canonical method of Chothia et al. However, the canonical method is limited to those loops where the key residues identified by Chothia are present. With the number of antibody structures currently available, it is not possible to classify CDR-H3 into canonical ensembles. Additionally, a small percentage of examples in the remaining CDRs do not match the current canonical classifications and the protein engineer may well wish to mutate the key residues, precluding the use of Chothia's method for modeling the resulting conformation. Thus the best approach appears to be to use Chothia's method (at least to model the backbone conformation) when the loop to be modeled is represented in the database of canonical structures. Any other loops, either unrepresented among the known canonicals (including CDR-H3), or where mutations have been made to the key residues, may then be modeled by the combined algorithm presented here.

Algorithms↗