Biomedical subjects

Dung M Vu

Publications and source records attributed to Dung M Vu.

7 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

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

Structural transformations in the dynamics of Michaelis complex formation in lactate dehydrogenase.

The dynamical nature of the binding of a substrate surrogate to lactate dehydrogenase is examined on the nanoseconds to milliseconds timescale by laser-induced temperature-jump relaxation spectroscopy. Fluorescence emission of the nicotinamide group of bound NADH is used to define the pathway and kinetics of substrate binding. Assignment of specific kinetic states and elucidation of their structures are accomplished using isotope edited infrared absorption spectroscopy. Such studies are poised to yield a detailed picture of the coupling of protein dynamics to function.

Binding Sites↗

PubMed2004

Experimental resolution of early steps in protein folding: testing molecular dynamics simulations.

Time-resolved Tyr fluorescence spectroscopy coupled with a laser-induced temperature-jump (T-jump) was employed to follow the folding relaxation dynamics of the B-domain of Staphylococcal protein A. The single Tyr is located in helix 1 (H1) and is a sensitive probe of the structure of this helix and the overall helical bundle structure. The results from this study were compared to those from a complementary infrared T-jump study on this protein [Vu, D. M.; Myers, J. K.; Oas, T. G.; Dyer, R. B. Biochemistry 2004, 43, 3582]. Both methods detect a microsecond process that follows the cooperative relaxation of the helical bundle core. However, a fast process (10-7 s) that follows the relaxation of the individual helices was observed only with the infrared probe. Thus, fast formation of H1 is not observed, but rather H1 forms in the microsecond phase, concomitantly with the docking to (and stabilization by) the other two helices to form the helical bundle structure. This observation validates the results of several previous molecular dynamics simulations that predict H1 formation only in the final assembly of the helix bundle.

Computer Simulation↗

PubMed2004

Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein.

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.

Amides↗

PubMed2003

NMR and temperature-jump measurements of de novo designed proteins demonstrate rapid folding in the absence of explicit selection for kinetics.

We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1. These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog. Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog. Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date.

Algorithms↗