Biomedical subjects

Scott H Brewer

Publications and source records attributed to Scott H Brewer.

10 recordsLinked to original sources

PubMed2007

Microfluidic flow-flash: method for investigating protein dynamics.

We report a new method, microfluidic flow-flash, for measuring protein reaction kinetics. The method couples a microscope imaging detection system with a microfluidic flow cell to reduce data acquisition times and sample consumption. This combination allows for the simultaneous collection of spectral and temporal information. The microfluidic flow cell design utilizes three-dimensional sheath flow to reduce sample dispersion and minimize sample consumption. The ability to alter the flow rates in the microfluidic flow cells allows a variety of time scales to be studied with submillisecond time resolution. The imaging detection system can be coupled with several spectroscopic probes including fluorescence and UV/visible absorbance spectroscopy. Here, we utilize the microfluidic flow-flash method to probe the kinetics of CO recombination or O2 binding to myoglobin after the laser-induced photolysis of CO from myoglobin by UV/visible absorbance spectral imaging.

Carbon Monoxide↗

PubMed2006

Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin.

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis were measured by femtosecond time-resolved absorption spectroscopy. Singular value decomposition (SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximately 9 ps and tau(12) approximately 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S = 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site >3 A from the heme iron with the greater barrier corresponding to a S = 5/2 spin state. The second SVD basis spectrum represents a time-dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximately 3 ps and tau(22) approximately 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP-NO and horse heart myoglobin (HHMbNO) or sperm whale myoglobin (SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild-type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP-NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP-NO also hold for the H64V mutant of SWMb (H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% (v/v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild-type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.

Amino Acid Substitution↗

PubMed2006

Testing bridge-mediated differences in dinuclear valence tautomeric behavior.

Two structurally characterized dinuclear valence tautomers are described. Cobalt ions are bridged by p- and m-phenylene units connected to 2,2'-bipyridines. X-ray crystal structures show that the molecules are in the [(Co(III))(Co(III))] forms at ca. 125 K, while spectroscopic studies show that both molecules can achieve the [(Co(II))(Co(II))] form above 400 K and confirm the [(Co(III))(Co(III))] form below 10 K. Magnetic susceptibility studies are also included. Our results highlight the necessity of studying both crystalline and amorphous samples to distinguish the effects of intrinsic electronic structure and intermolecular forces on valence tautomeric behavior.

Journal Article↗

PubMed2006

Time-resolved vibrational spectroscopy detects protein-based intermediates in the photosynthetic oxygen-evolving cycle.

Photosynthetic oxygen production by photosystem II (PSII) is responsible for the maintenance of aerobic life on earth. The production of oxygen occurs at the PSII oxygen-evolving complex (OEC), which contains a tetranuclear manganese (Mn) cluster. Photo-induced electron transfer events in the reaction center lead to the accumulation of oxidizing equivalents on the OEC. Four sequential photooxidation reactions are required for oxygen production. The oxidizing complex cycles among five oxidation states, called the S(n) states, where n refers to the number of oxidizing equivalents stored. Oxygen release occurs during the S(3)-to-S(0) transition from an unstable intermediate, known as the S(4) state. In this report, we present data providing evidence for the production of an intermediate during each S state transition. These protein-derived intermediates are produced on the microsecond to millisecond time scale and are detected by time-resolved vibrational spectroscopy on the microsecond time scale. Our results suggest that a protein-derived conformational change or proton transfer reaction precedes Mn redox reactions during the S(2)-to-S(3) and S(3)-to-S(0) transitions.

Electron Transport↗

PubMed2005

Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain.

Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 micros at 60.2 degrees C) and double phenylalanine mutant (3.01 micros at 49.9 degrees C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 mus at 49.9 degrees C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 micros compared with 6.97 micros for the WT at 49.9 degrees C.

Kinetics↗

PubMed2005

Probing BSA binding to citrate-coated gold nanoparticles and surfaces.

The interaction of bovine serum albumin (BSA) with gold colloids and surfaces was studied using zeta-potential and quartz crystal microbalance (QCM) measurements, respectively, to determine the surface charge and coverage. The combination of these two measurements suggests that BSA binding to gold nanoparticles and gold surfaces occurs by an electrostatic mechanism when citrate is present. The binding of BSA to bare gold is nearly two times greater than the binding of BSA to a citrate-coated gold surface, suggesting that protein spreading (denaturation) on the surface may occur followed by secondary protein binding. On the other hand, binding to citrate-coated gold surfaces can be fit to a Langmuir isotherm model to obtain a maximum surface coverage of (3.7 +/- 0.2) x 10(12) molecules/cm(2) and a binding constant of 1.0 +/- 0.3 microM(-1). The zeta-potential measurements show that the stabilization of colloids by BSA has a significant contribution from a steric mechanism because the colloids are stable, even at their isoelectric point (pI approximately 4.6). To be consistent with the observed phenomena, the electrostatic interactions between BSA and citrate must consist of salt-bridges, for example, of the carboxylate-ammonium type, between the citrate and the lysine on the protein surface. The data support the role of strong electrostatic binding but do not exclude contributions from steric or hydrophobic interactions with the surface adlayer.

Journal Article↗

PubMed2004

Characterization of single- and double-stranded DNA on gold surfaces.

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.

DNA↗

PubMed2004

Infrared detection of a phenylboronic acid terminated alkane thiol monolayer on gold surfaces.

Polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS) and infrared reflectance absorption spectroscopy (IRRAS) have been used to characterize the formation of a self-assembled monolayer of N-(3-dihydroxyborylphenyl)-11-mercaptoundecanamide) (abbreviated PBA) on a gold surface and the subsequent binding of various sugars to the PBA adlayer through the phenylboronic acid moiety to form a phenylboronate ester. Vibrationally resonant sum frequency generation (VR-SFG) spectroscopy confirmed the ordering of the substituted phenyl groups of the PBA adlayer on the gold surface. Solution FTIR spectra and density functional theory were used to confirm the identity of the observed vibrational modes on the gold surface of PBA with and without bound sugar. The detection of the binding of glucose on the gold surface was confirmed in part by the presence of a C-O stretching mode of glucose and the observed O-H stretching mode of glucose that is shifted in position relative to the O-H stretching mode of boronic acid. An IR marker mode was also observed at 1734 cm(-1) upon the binding of glucose. Additionally, changes in the peak profile of the B-O stretching band were observed upon binding, confirming formation of a phenylboronate ester on the gold surface. The binding of mannose and lactose were also detected primarily through the IR marker mode at approximately 1736 to 1742 cm(-1) depending on the identity of the bound sugar.

Alkanes↗

PubMed2004

Covalent attachment of a nickel nitrilotriacetic acid group to a germanium attenuated total reflectance element.

The surface of a germanium internal reflectance element (IRE) was modified to bind 6X-histidine (his)-tagged biomolecules. The step-by-step surface modification was monitored via single-pass attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR). Initially an adlayer of 7-octenyltrimethoxysilane (7-OTMS) was formed on the Ge crystal through the surface hydroxyl groups, which were produced via ozonolysis of the Ge surface. The vinyl moiety of 7-OTMS was oxidized to a carboxylic acid, which was activated by 1,1'-carbonydiimidazole (CDI) to produce a labile imidazole. The labile imidazole that resulted from the CDI coupling was then displaced by the primary amine of nitrilotriacetic acid (NTA). Nickel sulfate was added to the system, and it coordinated with the three carbonyl groups and the nitrogen on NTA, thus leaving the ability of Ni to coordinate with two adjacent histidine residues. Binding of his-tagged biotin to nickel nitrilotriacetic acid (Ni-NTA) was observed by ATR-FT-IR spectroscopy. The surface modification method presented in this paper had minimal nonspecific binding, the Ni-NTA surface was reusable if stored properly, and complete removal of the organic surface was achievable.

Journal Article↗

PubMed2003

Laser-induced temperature jump electrochemistry on gold nanoparticle-coated electrodes.

Laser-induced temperature jumps (LITJs) at gold nanoparticle-coated indium tin oxide (ITO) electrodes in contact with electrolyte solutions have been measured using temperature-sensitive redox probes and an infrared charge-coupled device. Upon irradiation with 532 nm light, interfacial temperature changes of ca. 20 degrees C were recorded for particle coverages of ca. 1 x 1010 cm-2. In the presence of a redox molecule, LITJ yields open-circuit photovoltages and photocurrents that are proportional to the number of particles on the surface. When ssDNA was used to chemisorb nanoparticles to the ITO surface, solution concentrations as low as 100 fM of target ssDNA-modified nanoparticles could be detected at the electrode surface.

DNA, Single-Stranded↗