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F J Stevens

Publications and source records attributed to F J Stevens.

At least 55 records · Page 3Linked to original sources
PubMed1995

Engineering a domain-locking disulfide into a bacterial malate dehydrogenase produces a redox-sensitive enzyme.

Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. We have modeled the tertiary structure of four of these light-activated enzymes, namely NADP-linked malate dehydrogenase, glyceraldehyde-3-P dehydrogenase, fructosebisphosphatase, and sedoheptulosebisphosphatase, and identified cysteines in each enzyme that be expected to form inactivating disulfide bonds (Li, D., F. J. Stevens, M. Schiffer, and L. E. Anderson, 1994. Biophys. J. 67:29-35). We have now converted two residues in the Escherichia coli NAD-linked malate dehydrogenase to cysteines and produced a redox-sensitive enzyme. Oxidation of domain-locking cysteine residues in the mutant enzyme clearly mimics dark inactivation of the redox-sensitive chloroplast dehydrogenase. This result is completely consistent with our proposed mechanism.

Base Sequence↗
PubMed1994

Solution-phase binding of monoclonal antibodies to bee venom phospholipase A2.

The binding of monoclonal anti-bee venom phospholipase A2 antibodies to their antigen was monitored by size-exclusion high performance liquid chromatography. As judged by this panel of six antibodies, honeybee venom phospholipase A2 contains five binding sites, three of which are completely independent epitopes. The study revealed that this PLA2 can accommodate three different antibodies simultaneously. The results demonstrate the utility of size-exclusion high performance liquid chromatography in epitope analyses, such as its ability to compare the relative expansiveness and conformational state of the epitopes and to enumerate the antibodies that the antigen can accommodate simultaneously. The data provide compelling evidence that one of the monoclonal antibodies, M5 (which activates the enzyme), recognizes a different conformation of phospholipase A2 than do the other antibodies. The results also demonstrate that different pairs of monoclonal antibodies differ in their predilection to form high molecular weight complexes with the antigen.

Animals↗
PubMed1994

Comparison of crystal structures of two homologous proteins: structural origin of altered domain interactions in immunoglobulin light-chain dimers.

The sequence and structure of a second human kappa 1 immunoglobulin light-chain variable domain, Wat, has been determined. The R-factor is 15.7% for 1.9-A data. One hundred and ninety-five water molecules were identified; 30 water molecules were located in identical positions in each of the monomers. Some of the water molecules are integral parts of the domains. This light chain is encoded by the same variable domain gene that encoded the previously characterized kappa I variable domain, Rei. Due to limited somatic mutation, the two highly homologous proteins differ in only 20 of the 108 residues. Wat crystallized in space group P6(4) while Rei crystallized in space group P6(1); in both crystals, the asymmetric unit was the noncovalent dimer. Although the basic domain structure is the same for both proteins, the relative positions of the domains within the two dimers differ. This difference is most likely accounted for by the replacement of Tyr36 in Rei by Phe in the Wat protein. Residue Tyr36 is part of the hydrogen-bonding network in the interface between the domains in Rei. Losing the hydrogen-bonding capability of residue 36 by replacement of Tyr by Phe alters the network of hydrogen bonds between the domains, resulting in a different domain-domain contact. The details of lattice contacts in the two crystals were compared. One type of contact that extends the beta-sheet of the individual domains was conserved, but because it involved different symmetry elements within the crystal, different crystal packing resulted. In the Wat crystal, one of the contacts shows an example of how a symmetrical binding site can "bind" an asymmetrical object. Further, the examination of the Wat crystal also illustrates how the different crystalline environments of the domains of the dimer results in different distributions of temperature factors for the residues within the domains.

Amino Acid Sequence↗
PubMed1994

Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition.

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.

Bence Jones Protein↗
PubMed1994

The hyperthermophilic glycolytic enzyme enolase in the archaeon, Pyrococcus furiosus: comparison with mesophilic enolases.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of > 90 degrees C and a pH optimum of 8.1. The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min. In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P. furiosus enolase was equally active in the presence of Mn2+. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids.

Amino Acid Sequence↗
PubMed1994

Mechanism of light modulation: identification of potential redox-sensitive cysteines distal to catalytic site in light-activated chloroplast enzymes.

Light-dependent reduction of target disulfides on certain chloroplast enzymes results in a change in activity. We have modeled the tertiary structure of four of these enzymes, namely NADP-linked glyceraldehyde-3-P dehydrogenase, NADP-linked malate dehydrogenase, sedoheptulose bisphosphatase, and fructose bisphosphatase. Models are based on x-ray crystal structures from non-plant species. Each of these enzymes consists of two domains connected by a hinge. Modeling suggests that oxidation of two crucial cysteines to cystine would restrict motion around the hinge in the two dehydrogenases and influence the conformation of the active site. The cysteine residues in the two phosphatases are located in a region known to be sensitive to allosteric modifiers and to be involved in mediating structural changes in mammalian and microbial fructose bisphosphatases. Apparently, the same region is involved in covalent modification of phosphatase activity in the chloroplast.

Amino Acid Sequence↗
PubMed1994

Dimerization of Bence Jones proteins: linking the rate of transcription from an Escherichia coli promoter to the association constant of REIV.

Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops. Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV). The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering. Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV. By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E. coli can be derived from REIV homodimer formation constant. The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E. coli chromosome. There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter. Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E. coli for immunoglobulin variants with altered association constants.

Bacterial Proteins↗
PubMed1992

The functions of tryptophan residues in membrane proteins.

Membrane proteins have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are not uniformly distributed through the membrane but are concentrated at the periplasmic side of the complex. In addition, Trp residues are not randomly aligned. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. These observations suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.

Hydrogen Bonding↗
PubMed1991

Effects of pH and calcium ion on self-association properties of two dimeric phospholipases A2.

The monomer-dimer equilibria of the dimeric phospholipases A2 from Crotalus atrox and Agkistrodon piscivorus piscivorus venoms were examined chromatographically as a function of pH and in the presence versus absence of the essential cofactor, calcium ion. At neutral pH without calcium, the subunits of both enzymes reequilibrated sufficiently slowly that dimer and monomer were separated by size exclusion chromatography. At pH 4.2 and lower, the dimers underwent rapid dissociation and reassociation, eluting as single broad peaks whose position as a function of applied protein concentration could be analyzed to determine association constants using an algorithm that estimates these values based on elution positions. Lowering the pH from 7.0 to 4.2 increased the self-association constant of the C. atrox enzyme by 1 order of magnitude and that of the A. p. piscivorus dimer by a factor of 3. Calcium ion, an essential cofactor of phospholipase A2, converted the kinetic behavior of the dimers at neutral pH from slow to virtually instantaneous on the time scale of the chromatography runs, 40 min. Calcium ion also altered the thermodynamic stability of the enzymes; the association constant of A. p. piscivorus phospholipase A2 in neutral pH buffer was reduced by approximately 2 orders of magnitude, whereas that of C. atrox was increased by a factor of 6. The structural basis for the disparate effects of calcium ion on these two acidic, dimeric venom phospholipases A2 is uncertain. This study illustrates the importance of calcium ion and pH on the solution behavior of the dimeric members of this class of enzymes.

Calcium↗
PubMed1989

Structure of a second crystal form of Bence-Jones protein Loc: strikingly different domain associations in two crystal forms of a single protein.

We have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-A resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding "pocket". The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion (Chang et al., 1985). The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry.

Bence Jones Protein↗
PubMed1989

Size-exclusion high-performance liquid chromatography in analysis of protein and peptide epitopes.

Size-exclusion HPLC provides direct observation of antibody-antigen interactions in free solution without chemical modification of either component. HPLC data collection systems are now a routine resource in both academic and industrial research laboratories. Monoclonal antibodies are, in principle, homogeneous reagents whose interactions with epitope are suitable for unambiguous quantitative characterization. HPLC-based methods, therefore, have the potential to contribute significantly to efficient quantitative characterization of monoclonal antibodies in studies directed to the fundamental physiological roles of these molecules as well as to the optimized selection and quality control of these reagents in biotechnical applications such as represented by the immunodiagnostic industry. In many cases, HPLC-based methods may represent the simplest and most rapid approach for evaluation of relative epitope specificities and affinity/kinetic characteristics of interaction.

Antigen-Antibody Complex↗
PubMed1989

Analysis of protein-protein interaction by simulation of small-zone size exclusion chromatography. Stochastic formulation of kinetic rate contributions to observed high-performance liquid chromatography elution characteristics.

High-performance liquid chromatography (HPLC) procedures provide size-exclusion chromatography with sufficient speed that the elution characteristics of mixtures of interacting macromolecules are potentially determined by the kinetics of association and dissociation. However, few studies have yet addressed the consequences of interaction kinetics on HPLC analyses or evaluated the potential application of HPLC methods for the qualitative and quantitative interpretation of macromolecular interaction kinetics. An earlier simulation of small-zone chromatography of interacting molecules (Stevens, F. J. 1986. Biochemistry. 25:981-993) has been modified to incorporate the effects of association/dissociation kinetics on elution behavior. The previous assumption of instantaneous equilibration has been replaced by explicit calculation of partial relaxation of complexed and free constituent mixtures during each iteration of the simulation. In addition, a stochastically based formulation has been introduced to determine a velocity probability distribution that emulates the partial intermixing of free and complexed pools during the iteration cycle. The simulation generates bimodal elution profiles representing stable complexed and free components of mixtures for which interaction is characterized by slow kinetics relative to chromatography run times. For mixtures with rapid kinetics, a single-asymmetric peak results. When tested with a large-zone sample such that a plateau of stable concentration is generated, the simulation reproduces previous characterizations based on evaluations of solute continuity equations. Therefore, HPLC may, in many cases be an appropriate basis for techniques by which to evaluate kinetic and affinity characteristics of interacting biomolecules.

Chromatography, Gel↗
PubMed1988

Analysis of immunoglobulin domain interactions. Evidence for a dominant role of salt bridges.

Available crystallographic data for homologous immunoglobulin constant domains were correlated with measured association constants for these domains. High correlation was found between the association constant and both the buried surface area (number of interdomain contacts) and the number of salt bridges formed in the interaction, whereas no correlation with the number of hydrogen bonds between domains was evident. The total free energy of binding, as determined from the association constant, was related to the number of contacts, hydrogen bonds and salt bridges found in the domain:domain interface by the crystallographic studies. These calculations yielded reasonable average energy terms for each interaction category.

Binding Sites↗
PubMed1988

Comparative developmental and phenotypic properties of altered hepatocyte foci and hepatic tumors in rats.

Previous investigations in this laboratory have provided evidence that histochemically detectable altered hepatocyte foci and hepatic tumors appearing in rats given a single neonatal treatment with a low dose of carcinogen followed by chronic dietary phenobarbital administration are developmentally independent. The present investigation further evaluates developmental relationships among these lesions. Altered hepatocyte foci were divided into two subclasses consisting of foci that were detectable by histochemical as well as by hematoxylin-eosin staining [designated hist(+)/morph(+) foci] and those foci that were detectable solely by histochemical staining [designated hist(+)/morph(-) foci]. The developmental and phenotypic properties of the hist(+)/morph(-) foci, hist(+)/morph(+) foci, and hepatic tumors were compared in rats initiated once neonatally with different doses of diethylnitrosamine and promoted with dietary phenobarbital from weaning. The morph(+) and morph(-) lesion subclasses were distinguishable on the basis of several developmental characteristics. Hist(+)/morph(+) foci were present at low frequency until at least 150 days after initiation. Although the development of hist(+)/morph(-) foci was essentially complete at that point, the rate of appearance of hist(+)/morph(+) increased significantly. The diethylnitrosamine dose response of the hist(+)/morph(+) foci followed the histochemical marker patterns of the tumor lesion class more closely than that of the hist(+)/morph(-) group. The rates of expression of the hist(+)/morph(+) foci increased with the increasing level of histochemical complexity, whereas the rates of expression of the hist(+)/morph(-) foci groups were inversely correlated to their complexity level. Although the average focus size or diameter in the hist(+)/morph(+) groups was greater than that of the hist(+)/morph(-) foci, the focus growth rates of morph(+) and morph(-) subsets matched for histochemical phenotype were comparable. The complexity level and individual marker distribution patterns of the hist(+)/morph(+) focus class were more similar to tumor patterns than to the distribution patterns of the hist(+)/morph(-) lesion class. The results suggest the following. (a) The development of lesion classes with successively greater deviation from normalcy does not occur via lineal progression from less to more deviated forms within a given lesion class. The three lesion classes appear to develop independently, with the developmental characteristics of each lesion class determined at the time of initiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Size-exclusion HPLC analysis of epitopes.

A size-exclusion chromatography method based on competition between intact antibodies and antigen-binding fragments (Fabs) is used to evaluate relative spatial location of epitopes recognized by monoclonal antibodies. If epitopes are spatially distinct, the presence of a potentially competitive Fab results in formation of complexes of higher molecular weight than those formed by antigen and intact IgG. Complexes of lower molecular weight are formed if the epitopes overlap, are identical, or if, upon formation of the antigen-antibody complex, conformational change occurs that results in loss of epitope. Size-exclusion chromatography of the relevant mixtures reveals these shifts in molecular weight distribution and, therefore, provides an unambiguous determination of spatially independent epitopes. In addition, chromatographic analysis of competition between IgG and its enzymatically prepared Fab is used to evaluate the dissociation rate constant of the antigen-antibody reaction in re-equilibrating mixtures.

Antibodies, Monoclonal↗