Biomedical subjects

James A Brozik

Publications and source records attributed to James A Brozik.

4 recordsLinked to original sources

PubMed2005

Framework model for DNA polymerases.

DNA polymerases are complex machines with both chemical and mechanical functions. Recent crystal structures, ensemble kinetics, and single-molecule investigations have helped to elucidate the main properties of several DNA polymerases, all of which share common structural elements and a common basic mechanism, despite wide variations in amino acid sequence. The framework model is intended to aid in the understanding of these common features (and differences). It defines a class of models that automatically incorporates most of what is known about DNA polymerases within a single theoretical structure so that it is easier to make comparisons between them and to generate detailed models for specific polymerases. The framework model has three main elements: (1) a set of four key variables that describe the important motions within the protein-DNA-nucleotide complex, (2) a complete set of conformational states for the protein-DNA-nucleotide system, and (3) an approximate potential energy surface that controls the motions and transition rates between states. As an example application, we use the general framework ideas to build a detailed model for the HIV reverse transcriptase that is consistent with existing data, and predicts force-velocity curves and stepping-statistics histograms that can be directly compared to experiment.

Animals↗

PubMed2004

Closing of the fingers domain generates motor forces in the HIV reverse transcriptase.

Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (approximately 15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.

Binding Sites↗

PubMed2004

Transient mixed-valence character of Re(I)(4)(CO)(12)(4,4'-bpy)(4)Cl(4).

This study addresses, in detail, the orbital nature and the extent of metal-metal communication in the lowest emitting triplet state of Re(4)(CO)(12)(4,4'-bpy)(4)Cl(4) (where 4,4'-bpy = 4,4'-bipyridine) as well as the symmetry of the lowest (3)MLCT manifold in comparison to that of the ground state. All spectral evidence points to (1). a (3)MLCT excited manifold localized between a single Re(I) corner and an adjacent bridging ligand, (2). a transient mixed-valence state that is completely localized between a single transiently oxidized Re center and the adjacent metals, and (3). a second-order charge transfer from a localized transiently reduced bridging ligand to the adjacent Re(I) center to which it is attached, effectively lowering its oxidation state. The orbital nature of the lowest (3)MLCT manifold is fully corroborated by a molecular orbital diagram derived from quantum chemical modeling studies, while the existence of the localization, localized mixed valency, and second-order charge transfer rely on spectral evidence alone. This work makes use of low-temperature time-resolved infrared (TRIR) techniques as well as a luminescence study. Many of the nuances of the luminescence and TRIR data interpretation are extracted from statistical analysis and quantum chemical modeling studies. The relative concentrations of the dominant conformers that exist for Re(4)(CO)(12)(4,4'-bpy)(4)Cl(4) have also been estimated from Boltzmann statistics.

Journal Article↗

PubMed2003

Non-localized ligand-to-metal charge transfer excited states in (Cp)2Ti(IV)(NCS)2.

The bent d(0) titanium metallocene (Cp)(2)Ti(NCS)(2) exhibits an intense phosphorescence from a ligand-to-metal charge transfer triplet excited state at 77 K in an organic glass substrate and a poly(methyl methacrylate) plastic substrate. Quantum chemical calculations and spectroscopic studies show that the orbital parentage of this triplet state arises from the promotion of an electron from an essentially nonbonding symmetry adapted pi molecular orbital located on the NCS(-) ligands to a d(z)2-(y)2 orbital located on the Ti metal. Standard infrared spectroscopy of (Cp)(2)Ti(NCS)(2) in its ground electronic state at 77 K reveals a pair of closely spaced absorptions at (2072 cm(-1), 2038 cm(-1))(glass) and (2055 cm(-1), 2015 cm(-1))(plastic) that are assigned, respectively, to the symmetric and antisymmetric CN stretching modes of the two coordinated NCS(-) ligands. Low-temperature (77 K) time-resolved infrared spectroscopy that accesses the phosphorescing triplet excited state on the ns time scale shows an IR bleach that is coincident with the two ground state CN stretching bands and an associated grow-in of a pair of new IR bands at slightly lower energies (2059 cm(-1), 2013 cm(-1))(glass) and (2049 cm(-1), 1996 cm(-1))(plastic) that are assigned, respectively, to the symmetric and antisymmetric CN stretches in the emitting triplet state. These transient IR bands decay with virtually identical lifetimes to those observed for the phosphorescence decays when measured under identical experimental conditions. Singular value decomposition analysis of the time-resolved infrared data shows that the observed transient IR features arise from the same electronic manifold as measured through luminescence studies. The close similarity between the ground state and excited-state CN stretching bands in (Cp)(2)Ti(NCS)(2) indicates that symmetry breaking does not occur in forming the charge-transfer triplet excited-state manifold; i.e., electron density is withdrawn from a delocalized pi MO spread across both NCS(-) ligands. Calculations at several levels of theory reveal a delocalized ligand-to-metal charge transfer excited triplet manifold. These calculations closely reproduce the relative intensity ratios and frequencies of the symmetric and antisymmetric transient infrared vibrations in the CN region. This study is the first time-resolved infrared investigation of a ligand-to-metal charge-transfer excited state and the first to be performed at cryogenic temperatures in thin-film organic glass and plastic substrates.

Journal Article↗