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Biomedical subjects

B L Barnett

Publications and source records attributed to B L Barnett.

18 recordsLinked to original sources
PubMed1999

Crystal structure of the stromelysin catalytic domain at 2.0 A resolution: inhibitor-induced conformational changes.

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83-255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three alpha-helices and one twisted, five-strand beta-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1' specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1' cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1' cavity, while in the complex, the P1' group of the inhibitor displaces the Tyr223 in order to fit into the S1' cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1' cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251-255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.

Allosteric Regulation↗
PubMed1997

Molecular modeling of the structures of human and rat pancreatic cholesterol esterases.

Structural models have been generated for rat and human cholesterol esterases by molecular modeling. For rat cholesterol esterase, three separate models were generated according to the following procedure: (1) the cholesterol esterase sequence was aligned with those of three template enzymes: Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase; (2) the X-ray structure coordinates of the three template enzymes were used to construct cholesterol esterase models by amino acid replacements of matched sequence positions and by making sequence insertions and deletions as required; (3) bad contracts in each of the cholesterol esterase models were relaxed by molecular dynamics and mechanics; (4) the three cholesterol esterase models were merged into one by arithmetic averaging of atomic coordinates; (5) Ramachandran analysis indicated that the model generated from the AChE template possessed the best set of phi/psi angles. Therefore, this model was subjected to molecular dynamics, with harmonic constraints imposed on the C(alpha) coordinates to drive them toward the coordinates of the averaged model. (6) Subsequent relaxation by molecular mechanics produced the final rat cholesterol esterase model. A model for human cholesterol esterase was produced by repeating steps 1-3 above, albeit with the rat cholesterol esterase model as the template. Hydrophobic and electrostatic analyses of the rat and human cholesterol esterase models suggest the structural origins of molecular recognition of hydrophobic substrates and interfaces, of charged interfaces, and of bile salt activators.

Animals↗
PubMed1996

Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity.

Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.

Adsorption↗
PubMed1996

Laundry performance of subtilisin proteases.

Effective laundry protease performance against susceptible stains depends upon both the enzyme itself and the environment in which it must work. In order to technically design superior laundry proteases, a model for protease's mechanism of action in detergents was developed which has been substantiated through-the-wash. While evaluation of this model and/or a given protease's effectiveness could be judged by a variety of methods, the utility of using visual wash performance comparisons, analytical, and stain characterization studies is described. Finally, data comparing the performance of wild type Subtilisin proteases with mutants designed via the projected model are given, demonstrating possible utility of the system.

Catalysis↗
PubMed1996

Free energy perturbation techniques applied to subtilisin BPN' stability.

The inability to predict the effect of specific mutations on protein stability has been an area of concern to researchers in the field of protein engineering. Small stabilization free energies (5 to 15 kcal/mol) distinguish the native and the denatured states of a protein, making the rational design of protein stability a difficult challenge. Free Energy Perturbation Technique (FEPT) appears to be a method that will be important for protein engineering to meet this challenge. Not only is it a method to evaluate potential sites for mutation prior to synthesis, it identifies important atomic contributions that are responsible for the free energy changes of interest. Accuracy and speed are the principal limitations of the technique, but the powerful combination of structure, energy, dynamics and solvent make the investment of time and effort very attractive. Our examples illustrate both the power and limitations of FEPT. Using the program CHARMm, FEPT has been applied to the well known stabilizing mutations--asparagine to Serine--at residue 218 in subtilisin BPN'. In analyzing the atomic contributions that result in the increase in stability, two mutations at residue 203 were chosen to test the predictive power of FEPT. Sometimes extraordinary measures must be undertaken to sample sufficient conformational space to achieve accurate FEPT results. However we believe the method will be invaluable in the development of rules for designing a more stable subtilisin BPN'.

Computer Simulation↗
PubMed1995

Recognizing borderline personality disorder in the family practice setting.

The first step in the management of borderline personality disorder is making the correct diagnosis. A clinical example illustrates symptoms of a patient with borderline personality disorder in a family practice setting. Major characteristics of borderline personality disorder include severe mood instability, fear of abandonment, chronic boredom, self-injury, unstable interpersonal relationships, "splitting," identity instability and borderline rage. Early diagnosis may help prevent potential management problems and possible doctor-patient conflicts.

Borderline Personality Disorder↗
PubMed1995

Directed evolution of a subtilisin with calcium-independent stability.

Extracellular proteases of the subtilisin-class depend upon calcium for stability. Calcium binding stabilizes these proteins in natural extracellular environments, but is an Achilles' heel in industrial environments which contain high concentrations of metal chelators. Here we direct the evolution of calcium-independent stability in subtilisin BPN'. By deleting the calcium binding loop from subtilisin, we initially destabilize the protein but create the potential to use new structural solutions for stabilization. Analysis of the structure and stability of the loop-deleted prototype followed by directed mutagenesis and selection for increased stability resulted in a subtilisin mutant with native-like proteolytic activity but 1000-times greater stability in strongly chelating conditions.

Biological Evolution↗
PubMed1990

Gene 5 protein-DNA complex: modeling binding interactions.

A helical (not toroidal) complex consisting of eight gene 5 protein dimers per turn is proposed for the extension of DNA from dimer to dimer using known bond length constraints, postulated protein-nucleic acid interactions (determined from NMR and chemical modification studies), other physical properties of the complex, and data from electron micrographs. The binding channel has been dictated by these known parameters and the relative ease of geometrically fitting these constituents. This channel is different from that previously reported by other modelers. The channel lies underneath the long arm "claw-like" extension of the monomer, so that it rests inside the outer surface of the protein complex. An explanation is proposed for the two binding modes, n = 4 (the predominate mode) and n = 3, based on the weak binding interaction of Tyrosine 34. Also, the site of the less mobile nucleic acid base as reported from ESR studies (S.-C. Kao, E.V. Bobst, G.T. Pauly and A.M. Bobst, J. Biom. Struc. Dyn. 3,261 (1985)) is postulated as involving the fourth nucleotide, and this particular base is stacked between Tyrosine 34 and Phenylalanine 73'.

Bacteriophages↗
PubMed1990

Effects of engineered salt bridges on the stability of subtilisin BPN'.

Variants designed using PROTEUS have been produced in an attempt to engineer stabilizing salt bridges into subtilisin BPN'. All the mutants constructed by site-directed mutagenesis were secreted by Bacillus subtilis, except L75K. Q19E, expressed as a single variant and also in a double variant, Q19E/Q271E, appears to form a stabilizing salt bridge based on X-ray crystal structure determination and differential scanning calorimeter measurements. Although the double mutant was found to be less thermodynamically stable than the wild-type, it did exhibit an autolytic stability about two-fold greater under hydrophobic conditions. Four variants, A98K, S89E, V26R and L235R, were found to be nearly identical to wild-type in thermal stability, indicative of stable structures without evidence of salt bridge formation. Variants Q271E, V51K and T164R led to structures that resulted in varying degrees of thermodynamic and autolytic instability. A computer-modeling analysis of the PROTEUS predictions reveals that the low percentage of salt bridge formation is probably due to an overly simplistic electrostatic model, which does not account for the geometry of the pairwise interactions.

Bacillus subtilis↗
PubMed1980

Tc-99m HMDP (hydroxymethylene diphosphonate): a radiopharmaceutical for skeletal and acute myocardial infarct imaging. I. Synthesis and distribution in animals.

Technetium-99m hydroxymethylene diphosphonate (Tc-99M HMDP) is a new diphosphonate skeletal imaging agent. Animal studies show that Tc-99m HMDP has a higher uptake on bone and a more rapid clearance from the blood than any of the three technetium-labeled bone imaging agents in current use: Tc-99m methylene diphosphonate (DMP), Tc-99 (1-hydroxyethylidene) diphosphonate (HEDP), and Tc-99m pyrophosphate (PPi). On the basis of these animal studies, Tc-99m HMDP is a highly promising candidate for skeletal imaging.

Acute Disease↗
PubMed1980

Tc-99m HMDP (hydroxymethylene diphosphonate): a radiopharmaceutical for skeletal and acute myocardial infarct imaging. II. Comparison of Tc-99m hydroxymethylene diphosphonate (HMDP) with other technetium-labeled bone-imaging agents in a canine model.

Technetium-99m hydroxymethylene diphosphate (Tc-HMDP) was compared with the two other diphosphonates (Tc-MDP and Tc-HEDP) and Tc-99m pyrophosphate (Tc-PPi) in a canine model of acute myocardial infarction. The TC-HMDP showed higher uptake in infarcted myocardium than the other two diphosphonates, and uptake equivalent to that of Tc-PPi.

Animals↗
PubMed1978

Basic science in a predoctoral family practice curriculum.

A course in applied basic science was designed with topic material organized according to anatomic body regions. Details of the diagnostic method were explained early in the course, and clinical procedures for data gathering and problem analyzing were followed while the significance of basic science knowledge in dealing with clinical situations was described. A collection of 35mm slides constituted the focal point of the course. The authors conducted the course together and an atmosphere of intellectual honesty was developed through open discussion between faculty and students. Student curiosity was respected and rewarded. Summaries of the discussions were prepared retrospectively by the faculty instructors for review gy the students. This experience proved that family physicians can demonstrate effectively the relevance of basic science to clinical medicine.

Curriculum↗
PubMed1977

A new look at the consultation continuum.

The purpose of this paper is to bring into sharp focus the intricate and vital linkages among the active participants in the consultation process (Figure 1). For too long the profession has been locked into a ritualistic, buck-passing processing frequently resulting in unorganized efforts on behalf of objects rather than subjects. The essential overriding concern then could well be represented by the center diagram (the patient and his family) and the supporting persons - communicating before, during, and after the consultation - completing a process which could bring about improvement in the quality of life for the patient, the referring physician, and the family. Through the added efforts to give feedback to the specialist we could conceivably improve the consultant's quality of life too.

Family Practice↗
PubMed1976

The post-suicide family and the family physician.

It is estimated that there are 750,000 people each year who are intimately affected by suicide. Prominent among these are the family survivors and their family physician. This paper offers a time frame which divides the period following the suicide into three phases: Immediate (the first ten days after the suicide); Intermediate (after the first ten days through the first year); and Extended (from the first year until restitution occurs). It identifies the chief emotional reactions which occur in each phase, explores their psychodynamic origins, and proposes suggestions for appropriate management during each of the three periods. The goal of this plan of management is to enable the family physician to function in a supportive emphatic, and restorative manner for the post-suicide family.

Depression↗