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R J Leatherbarrow

Publications and source records attributed to R J Leatherbarrow.

17 recordsLinked to original sources
PubMed1992

Imaging of proteins by scanning tunnelling microscopy.

Scanning tunnelling microscopy has been used to examine the structure of proteins deposited on a graphite surface. Three molecules have been studied; immunoglobulin G (IgG), Complement component 1q (C1q) and ATP-citrate lyase (ACL). The images show IgG as a tri-lobed molecule, consistent with the known 3D structure as determined by X-ray crystallography. The C1q images differ from the well known "tulip bunch" model derived by electron microscopy, but are consistent with the model if it is assumed that the six globular heads have aggregated. Molecules of ACL are visible as discrete units, with some hints of substructure. These results highlight the potential of STM in studying protein structures, but also illustrate the difficulties of interpreting micrographs of proteins whose structure is currently unknown.

Complement C1q↗
PubMed1991

Design of a small peptide-based proteinase inhibitor by modeling the active-site region of barley chymotrypsin inhibitor 2.

A synthetic peptide-based proteinase inhibitor was constructed by modeling the regions responsible for inhibition in barley chymotrypsin inhibitor 2 (CI-2). The 18-residue peptide was designed by molecular modeling, based on the crystal structure of CI-2. The amino acid sequences that interact with the proteinase were preserved, as well as residues that maintain the structure of the inhibitory loop. A disulfide bridge was introduced to force the peptide to adopt a cyclic structure. Kinetic studies on binding of the cyclic peptide to subtilisin BPN', subtilisin Carlsberg, chymotrypsin, and pancreatic elastase show that the cyclic peptide retains both the inhibition properties, the kinetic mechanism, and the specificity of the original protein inhibitor. Formation of a cyclic structure was found to be essential, and activity was abolished by reduction of the disulfide. As with CI-2, tightest binding is found to subtilisin BPN', where the Ki value for the cyclic peptide was 28 x 10(-12) M, compared with 29 x 10(-12)M for CI-2 under identical conditions. This remarkable result shows that it is possible to use a short synthetic peptide to model the molecular recognition properties of the intact protein, in this case obtaining full functionality with just 18 residues instead of 83 for CI-2.

Amino Acid Sequence↗
PubMed1991

Structure of immunoglobulin G by scanning tunnelling microscopy.

Scanning tunnelling microscopy (STM) has been used to examine the shape of individual immunoglobulin G (IgG) molecules deposited onto a graphite surface. IgG was chosen for this study as it has a well-characterized and distinctive three-dimensional structure. The micrographs clearly reveal the IgG molecule as trilobed, corresponding with the known structural organization of IgG. Comparison of these images with the structure of IgG determined by X-ray crystallography shows that the STM images are consistent with the crystal structure. This illustrates that STM is a valuable technique for examining protein structure, allowing rapid determination of the overall molecular shape that is consistent with more established techniques.

Immunoglobulin G↗
PubMed1990

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing.

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.

Adrenal Glands↗
PubMed1990

Role of arginine 67 in the stabilization of chymotrypsin inhibitor 2: examination of amide proton exchange rates and denaturation thermodynamics of an engineered protein.

We have examined the contribution to protein stability of an interaction involving a charged hydrogen bond from an arginyl side chain (Arg67) in the serine proteinase inhibitor chymotrypsin inhibitor 2 (CI-2), by replacing this side chain with an alanyl residue by protein engineering. Using nuclear magnetic resonance spectroscopy (NMR), we have examined the effect of this mutation on the hydrogen-deuterium exchange rates of several backbone amide protons in the native and engineered proteins at 50 degrees C. These exchange rates provide a localized probe at multiple discrete sites throughout the protein and from comparison of native and mutant exchange rates allow calculation of the difference in free energy of exchange (delta delta Gex) resulting from the mutation. The results show that for the majority of amides observed this mutation results in delta delta Gex of ca. 1.7 kcal mol-1 over the whole CI-2 molecule. However, for two relatively exposed amide protons the exchange rates are found to be far less perturbed, implying that local unfolding mechanisms predominate for these protons. Direct measurement of the stability of both proteins to denaturation by guanidinum hydrochloride shows that the interaction contributes 1.4 kcal mol-1 to the stability of the molecule. This value is comparable to those obtained from the NMR exchange measurements and indicates that the exchange processes reflect the differences in stability between the native and mutant proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1990

Metal ion dependence of phosphorothioate ATP analogues in the Bacillus stearothermophilus tyrosyl-tRNA synthetase reaction.

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Use of nonlinear regression to analyze enzyme kinetic data: application to situations of substrate contamination and background subtraction.

In a recent publication, A. Lundin, P. Arner, and J. Hellmér [Anal. Biochem. 177, 125-131 (1989)] describe a method whereby kinetic substrate assays can be performed when the assay mixture includes a significant contaminating levels of substrate. Their method requires various rearrangements of the data, and involves three separate linear regression calculations. We show how the same data may be analyzed directly, and far more simply, by nonlinear regression. Unlike the linear regression method, nonlinear regression allows direct calculation of the actual values for Km, Vmax, and the concentration of contaminating substrate (as well as estimates of their standard errors); the former method gives only apparent values. The nonlinear regression technique is also statistically a more valid means of analysis, as the rearrangements required to give linearized equations will considerably distort the error distribution and render simple unweighted linear regression inappropriate. The ease of incorporating extra parameters into standard equations when nonlinear regression is used is further illustrated by fitting enzyme reaction data which describe a first-order process when a significant nonspecific background is present. For this equation no simple rearranged linear plot is possible, but nonlinear regression is easily applied to determine the kinetic parameters.

Enzymes↗
PubMed1990

Molecular cloning, sequencing and expression of cytochrome c2 from Rhodospirillum rubrum.

Cytochrome c2 (Mr 12,840) of the purple photosynthetic bacterium Rhodospirillum rubrum functions as a mobile electron carrier in the cyclic photosynthetic electron-transport system of this organism. It acts as the electron donor to photochemically oxidized reaction centres and is reduced in turn by electrons from the cytochrome bc1 complex. By using synthetic oligonucleotides based on the known amino acid sequence of the protein, the structural gene (cycA) has been identified and isolated. DNA sequence analysis indicates the presence of a typical prokaryotic 23-residue signal sequence, suggesting that the protein is synthesized as a precursor which is processed during its secretion into the periplasm. Evidence is presented for the production of assembled cytochrome c2 in Escherichia coli, but recombinants grow poorly and are unstable, suggesting toxicity of the gene product in this organism.

Amino Acid Sequence↗
PubMed1990

Using linear and non-linear regression to fit biochemical data.

For biochemists or chemists the most common form of data analysis is likely to be regression analysis. This is a technique to find the 'best' values for various experimental parameters; defined as those values which, when used in an appropriate equation, result in the minimum deviation of the calculated results from the experimental data. Despite the widespread application of regression analysis, the basis of the technique and the underlying assumptions are often poorly understood or appreciated. This article describes the basics of linear and non-linear regression, the role of 'weighting' and the potential pitfalls of such analyses.

Biochemical Phenomena↗
PubMed1987

Investigation of transition-state stabilization by residues histidine-45 and threonine-40 in the tyrosyl-tRNA synthetase.

We have analyzed various mutations involving residues Thr-40 and His-45 in the tyrosyl-tRNA synthetase of Bacillus stearothermophilus. The utilization of binding energy in catalysis of tyrosyl adenylate formation from tyrosine and ATP was determined from the free energy profiles for the mutant enzymes. Our results confirm that the side chains of Thr-40 and His-45 provide a binding site for the pyrophosphoryl portion of the transition state of this reaction and for pyrophosphate in the reverse reaction. Deletion of these side chains destabilizes the transition-state by 4.9 and 4.1 kcal mol-1, respectively, consistent with a charged hydrogen-bonding interaction. To examine the role of His-45 further, we constructed the potentially conservative mutations His----Gln-45 and His----Asn-45. Both mutant enzymes are debilitated compared with the native enzyme. The His----Gln-45 enzyme is more active than enzyme in which the complete side chain is deleted (His----Ala-45), and so in this location glutamine is a semiconservative replacement. In contrast, the His----Asn-45 mutation is significantly worse than simple deletion of the side chain, indicating that asparagine at this position causes active destabilization of the transition state compared to His----Ala-45. The amide-NH2 of glutamine may be considered stereochemically equivalent to the epsilon-NH of histidine whereas the amide-NH2 of asparagine is comparable to the delta-NH of histidine. The results suggest that the epsilon-NH rather than the delta-NH group of His-45 is involved in the transition-state stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1987

Structure-activity relationships in engineered proteins: analysis of use of binding energy by linear free energy relationships.

The activity of mutant enzymes can be analyzed quantitatively by structure-activity relationships in a manner analogous to Brønsted or Hammett plots for simple organic reactions. The slopes of such plots, the beta values, indicate for the enzymatic reactions the fraction of the overall binding energy used in stabilizing particular complexes. In particular, information can be derived about the interactions between the enzyme and the transition state. The activities of many mutant tyrosyl-tRNA synthetases fit well simple linear free energy relationships. The formation of enzyme-bound tyrosyl adenylate (E.Tyr-AMP) from enzyme-bound tyrosine and ATP (E.Tyr-ATP) results in an increase in binding energy between the enzyme and the side chain of tyrosine and the ribose ring of ATP. Linear free energy plots of enzymes mutated in these positions give the fraction of the binding energy change that occurs on formation of the transition state for the chemical reaction and the various complexes. It is shown that groups that specifically stabilize the transition state of the reaction are characterized by beta values much greater than 1. This is found for residues that bind the gamma-phosphate of ATP (Thr-40 and His-45) and have previously been postulated to be involved in transition-state stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1985

Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor.

Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events.

Animals↗
PubMed1985

Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering.

The principal catalytic factor in the activation of tyrosine by the tyrosyl-tRNA synthetase is found to be improved binding of ATP in the transition state. The activation reaction involves the attack of the tyrosyl carboxylate on the alpha-phosphate group of ATP to generate a pentacoordinate transition state. Model building of this complex located a binding site for the gamma-phosphate group of ATP, consisting of hydrogen bonds with the side chains of Thr-40 and His-45. Removal of these groups by protein engineering shows that they contribute no binding energy with unreacted ATP but put all of their binding energy into stabilizing the [tyrosine-ATP] transition state [the mutant tyrosyl-tRNA synthetase (Thr-40----Ala-40; His-45----Gly-45) has the rate of formation of tyrosyl adenylate lowered by 3.2 X 10(5) but KS for ATP is lowered by only a factor of 5]. The side chains of these residues also provide a binding site for pyrophosphate in the reverse reaction. Thus, catalysis is accomplished by stabilization of the transition state by improved binding of a group on the substrate that is distant from the seat of reaction.

Adenosine Triphosphate↗
PubMedDate not supplied

Protein engineering.

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Carboxypeptidases↗