Biomedical subjects

S A McCallum

Publications and source records attributed to S A McCallum.

6 recordsLinked to original sources

PubMed2000

Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase.

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.

Binding Sites↗

PubMed1999

Solution structure of the carboxyl terminus of a human class Mu glutathione S-transferase: NMR assignment strategies in large proteins.

Strategies to obtain the NMR assignments for the HN, N, CO, Calpha and Cbeta resonance frequencies for the human class mu glutathione-S-transferase GSTM2-2 are reported. These assignments were obtained with deuterated protein using a combination of scalar and dipolar connectivities and various specific labeling schemes. The large size of this protein (55 kDa, homodimer) necessitated the development of a novel pulse sequence and specific labeling strategies. These aided in the identification of residue type and were essential components in determining sequence specific assignments. These assignments were utilized in this study to characterize the structure and dynamics of the carboxy-terminal residues in the unliganded protein. Previous crystallographic studies of this enzyme in complex with glutathione suggested that this region may be disordered, and that this disorder may be essential for catalysis. Furthermore, in the related class alpha protein extensive changes in conformation of the C terminus are observed upon ligand binding. On the basis of the results presented here, the time-averaged conformation of the carboxyl terminus of unliganded GSTM2-2 is similar to that seen in the crystal structure. NOE patterns and 1H-15N heteronuclear nuclear Overhauser enhancements suggest that this region of the enzyme does not undergo motion on a rapid time scale.

Amino Acid Sequence↗

PubMed1999

A J(CH)-modulated 2D (HACACO)NH pulse scheme for quantitative measurement of (13)C(alpha)-(1)H(alpha) couplings in (15)N, (13)C-labeled proteins.

A triple-resonance pulse sequence is presented for the quantitative measurement of (1)H(alpha)-(13)C(alpha) single-bond couplings in (15)N, (13)C uniformly labeled proteins. This (1)J(CH)-modulated (HACACO)NH experiment yields (1)H(N)-(15)N-correlated 2D spectra in which the amplitude of each peak is modulated by the (1)H(alpha)-(13)C(alpha) J coupling of the preceding residue, (i - 1). The experiment is demonstrated on a 1.0 mM sample of Rho130, the 15-kDa RNA binding domain of E. coli Rho factor. The average error in the measured coupling constants was less than 0.8%. This sequence allows the measurement of the (1)J(CH )couplings from a proton-nitrogen HSQC without the need for assigning the H(alpha) and C(alpha) resonances.

Carbon↗

PubMed1998

Unambiguous correlations of backbone amide and aliphatic gamma resonances in deuterated proteins.

Two 3D NMR pulse sequences that correlate aliphatic gamma carbon resonance frequencies to amide proton and nitrogen chemical shifts in perdeuterated proteins are presented. The HN(COCACB)CG provides only interresidue connectivities (NH(i) and Cgamma(i-1)) while the HN(CACB)CG detects both the inter- and intraresidue (NH(i) and Cgamma(i) or Cgamma(i-1)) correlations. These two experiments are useful for sequential assignments and the identification of residue type from the Cgamma shifts. Spectra acquired on a perdeuterated 53-kDa protein illustrate the sensitivity and utility of these experiments.

Amides↗

PubMed1987

The EcoR V restriction endonuclease.

Type II restriction endonucleases have attracted attention for two main reasons: firstly, their many applications in the dissection of DNA and in the construction of novel DNA molecules; secondly, as systems for studying the interactions of proteins with specific DNA sequences. With respect to the latter, the EcoR I restriction endonuclease has been examined in greater depth than any other type II enzyme [1-3]. However, the EcoR I enzyme has a major disadvantage as a system for studying DNA-protein interactions: the protein has a remarkably low solubility. The solutions in which EcoR I shows maximal activity, and also affinity for its recognition site, are saturated at less than 0.5 microM of this protein [4]. Consequently, many techniques that have been developed to study protein-ligand interactions but which require high concentrations of the protein in solution, such as NMR spectroscopy, cannot be used on EcoR I. But this drawback does not apply to all type II restriction enzymes. A different enzyme, the EcoR V restriction endonuclease [5-7], has special advantages as a system for studying DNA-protein interactions. In particular, this is the only type II restriction enzyme (apart from EcoR I [3]) for which crystals of the protein have been reported [7].

Cloning, Molecular↗

PubMed1986

Relaxed specificity of the EcoRV restriction endonuclease.

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.

Base Sequence↗