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[super 13]C[double bond][super 18]O isotope-edited infrared spectroscopy is used to examine the ability to resolve protein folding dynamics at the level of a single residue. The results have shown that a single [super 13]C[double bond][super 18]O label can be detected in a residue protein and that the signal-to-noise is enough for probing both equilibrium thermal unfolding and time-resolved folding 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 [O.sub.2] binding to myogiobin after the laser-induced photolysis of CO from myogiobin by UV/visible absorbance spectral imaging.

Beta-hairpins constitute an important class of connecting protein secondary structures. A simple framework is provided by peptide models for exploring the molecular details of the formation of beta-hairpin structures.

Research has been conducted on heme-copper oxidases. The covalently linked structure of this oxidase has been investigated via the use of infrared spectroscopy, synthetic model studies, normal mode calculations and isotopic substitution on tyrosine and histidine, and the results are reported.

Research has been conducted on the key residue motions at the substrate binding site of lactate dehydrogenase. Results indicate that after the formation of the productive protein/substrate complex the binding pocket is rigid with very little motion apart from the mobile loop and that the loop opening involves substrate movement out of the binding pocket.

Research has been conducted on the heme-copper respiratory oxidases. The redox-linked proton-pumping mechanism of these oxidases have been investigated via the use of cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared spectroscopy and the results are discussed.

Research has been conducted on the Ca (super)2+ -binding to the regulatory component of calcineurin. The use of the site-directed mutagenesis, flow dialysis and Fourier transform infrared spectroscopy to study this binding ia described.d

Results demonstrate that water is the axial ligand of the deoxy heme iron in the H93G proximal cavity myoglobin mutant. Data show that the CO adduct of the protein is a six-coordinate with a histidine trans ligand and it functions as a conformationally driven ligand switch.

Results demonstrate that the core formation in apomyoglobin follows robust dynamics comprising multiple folding pathways. The E or acid-destabilized form is a heterogeneous structure with a hydrophobic core composed of 30-40 residues at the intersection of AGH helices with traces of secondary structure.

The structure of the acid- and salt-destabilized E and I forms of the horse heart apomyoglobin represent the early structural configurations of this largely helical protein. Both forms exhibit AGH core but differ in melting thermodynamics. The destabilized E form converts to alpha-helix in the I form.

Mutagenesis and kinetic analysis reveal that in the wild-type human flap endonuclease-1, Asp181 residue mediates substrate catalysis. Data point out that the residue binds to the magnesium cofactor, which there upon cleaves the flap DNA.

The time-resolved infrared spectrum of [Ru(phen)3]2+* in the fingerprint region was examined. The spectra included information about both chromophoric and ancillary ligands since the experiment is independent of the requirement of resonance enhancement. Data gathered provided evidence for the localized description of [RuIII(phen)3]3+* on the 100 ns time scale. Results also aided in the interpretation of the time-resolved resonance Raman spectrum of the ligand and gave insight into electronic distribution in the excited state.

Vibrational data on deoxyferrous and ferrous-CO derivatives of the proximal histidine ligands in Notomastus lobatus chloroperoxidase (NCPO) and Amphitrite ornata dehaloperoxidase (DHP) are similar to data reported for neutral histidine/imidazole-ligated globins and heme complexes. This indicates that the proximal histidine ligands of NCPO and DHP do not have a special catalytic role in the two enzymes' mechanisms of action. It is possible that DHP and NCPO rely on another distal mechanism to generate the oxoferryl intermediate.

The bridging ligand 2,2':3',2'':6'',2'''-quaterpyridine was observed to have two inequivalent metal binding sites (A and B) which perturb the metal-to-ligand charge transfer (MLCT) energy levels of the attached Re(I) or Ru(II). Time-resolved infrared difference spectra of ((bpy)2Ru(II)ABRe(I)(CO)3Cl)2+ were in agreement with a lowest lying Ru(III)-AB*- MLCT state, while spectra of ((CO)3ClRe(I)ABRu(II)(bpy)2)2+ indicated the presence of both Re(II)-AB*- and Ru(III)-AB*- MLCT excited states.

Most experimental studies on protein folding dynamics have been limited to timescales of 1 ms or more. It is clear that many phenomena necessary for folding of proteins occur in a much shorter time than that. Formation of tertiary and secondary structures can take place on microsecond and nanosecond timescales, respectively. The fast events are required and dominate at times, but with only a few exceptions the study of these events experimentally has been possible only lately. New approaches can start and monitor the fast events with temporal resolution going down to picoseconds. Some important results for folding of some globular proteins and polypeptide models are now available.

Time-resolved infrared spectroscopy was utilized to investigate the mechanism governing the folding dynamics for the 'I' form of apomyoglobin (apoMb-I). Results have shown that the structure of apoMb-I was kinetically and thermodynamically heterogeneous comprising of AGH core. The stability of the core was determined by the presence of native interhelical contacts and solvated helical portions without interhelical contacts. The unfolding time was dependent on the stability of the helical segment.

A study was conducted on connectivity between CuB and glutamic acid 286 (Glu286) in cytochrome bo3 from Escherichia coli. The findings indicate an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. Moreover, this connectivity, which may be due to a chain of bound water molecules, may be of interest in the context of the proton pump mechanism of the heme-copper oxidases.

Metal-to-ligand charge transfer through time-resolved infrared (TRIR) was demonstrated in a study conducted on [Ru(bpy)(sub3)](super2+) and [Re(bpy)(CO)(sub3)(4-Etpy)](super+) (bpy is 2,2'-bipyridine) and (4-Etpy is 4-ethylpyridine). TRIR was then used in the fingerprint region from 1400 to 1625 cm(super-1) for transition metal complex at their excited states. Findings provided significant insight for electronic and molecular structure in the excited state, which focused on the directions and magnitudes of the infrared shifts.

A synthetic myoglobin was created to study the formation of six-coordinate complexes in the presence of nitric oxide. When nitric oxide binds with the heme protein, it typically weakens the nearest bond between heme and iron. In contrast, the synthetic myoglobin contains imidazole that functions as a ligand for iron. The presence of nitric oxide breaks this bond to form a five-coordinate complex. The six-coordinate complex can only be formed in the presence of excess imidazole, which shows that it has a lower binding constant than nitric oxide for the heme.

The dynamics of protein folding was observed in a small 21-residue alpha-helical alanine-based peptide. The rate of protein unfolding during helix melting in this peptide was generated using laser-induced temperature jump kinetics and probed using time-resolved infrared spectroscopy. The peptide exhibited fast unfolding kinetics within 20 nanoseconds with a time constant of 160 (+/-) 60 nanosecond at 28 degrees Celsius when treated with a laser-induced temperature jump of 18 degrees Celsius. The folding rate constant of this peptide was determined during helix formation at 28 degrees Celsius.

Thermus thermophilus bacteria yield cyano and unliganded derivatives of cytochrome ba3, and the process is analyzed by resonance Raman, ultraviolet and electroparamagnetic resonance spectroscopies. Cytochrome ba3 is an isolated, unliganded enzyme with structural similarities to the bovine cytochrome aa3.

Metal to ligand charge-transfer excitation occurs in the chromophore-quencher complexes cis-((bpy)2Os(II)(CO)(MQ+))3+, as indicated by transient resonance Raman spectra. The monoquant ligand is indicated by the transient spectra of the complex. A coplanar geometry is exhibited by the ligand and two pyridyl rings which are delocalized to the excited state.

Picosecond time-resolved spectroscopic studies on the reaction steps following photodissociation of carbon monoxide (CO) from cytochrome c oxidase's a3-CuB site reveal that CO transfers completely and the CuBCO complex forms within one picosecond. CO which is bound to a3 has an orientation angle of 20 degrees to the normal. The CO dipole has an orientation of about 55 degrees of the heme normal on binding with CuB.

An examination of the photochemistry of flash-induced photodissociation and the rejoining of carbon monoxide in cytochrome aa3-CO reveals that the secretion of ligand L from CuB may be caused by the picosecond association of photodissociated CO with CuB. During the attachment of various tiny molecules, especially O2 at the active region, 'ligand shuttle' may take place.

Time-resolved infrared spectroscopy (TRIR) was used to examine alterations in protein conformations combined with carbon monoxide's ligation interactions, in myoglobin (Mb). Alterations in the secondary structure of proteins connected with the global easing of the protein helices after photodissociation of CO are reasons for TRIR variation characteristics of Mb.

Electron transfer vibrational excitation and relaxation in the mixed-valence transition-metal dimer ((NC)5MIICNMIII(NH3)5)- (M = Ru, Os) were studied using picosecond infrared spectroscopy. Excited state redox isomer ((NC)5MIIICNMII(NH3)5)- is developed upon optical excitation into the metal-to-metal charge transfer bond. The ground state is developed again after back electron transfer. Massive quantities of energy are introduced into the terminal MC triple bonded to N stretching mode, upon the switching back to the ground electronic state.

The photophysical and spectroscopic properties of polynuclear complexes with cyano-bridged ruthenium polypyridine units are studied to investigate the extent of electronic interaction between the metal centers of these systems. A comparison of the infrared spectra of the mixed-valence forms of these polynuclear complexes with those of the mixed-valence ions of the type (M-NC-M'(bpy)2-CN-M)(super 6+/4+) and (NC-M'(bpy)2-CN-M)(super 3+/2+), where M = (Ru(NH3)5)(super 2+/3+) and M' = RuII, OsII and ReI, is also undertaken.

A discussion of time-resolved approaches to the characterization of the dynamical nature of the enzymatic catalysis and their application to specific enzyme systems is presented. The use of a trigger method to initiate chemical or structural changes in a system, combined with a structural probe which follows the evolution of the system in time is the essential feature of time-resolved studies.

The cold-denatured state of a beta-hairpin forming peptide, MrH3a, in 8% hexafluoro-2-propanol (HEIP) and the dynamics of its refolding following a laser-induced T-jump, beta-Hairpins constitute an important class of protein structural elements. Characterization of MrH3a using NMR, CD and IR spectroscopies reveals residual structure in the cold-denatured state, in contrast with the highly disordered heat-denatured state.

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A is carried out to identify the folding and unfolding dynamics of this small three-helix bundle protein. Investigation of data obtained provides significant understanding of the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.

Molecular dynamics simulations of small, fast folding proteins are beginning to display the details, but validation of these simulations required some experimental tests. The predications of some MD simulations that the formation of M1 occurs very late in the folding of this protein, together with the formation of the tertiary helical bundle is confirmed in the result.

Research is presented demonstraing the importance of hydrophobic interactions in the secondary structure formation of peptides. The hydrophobically driven beta hairpin formation is examined using FT-IR and 13C NMR.

The excited-state spectra in three polypyridyl complexes of rhenium(I) were determined. Time-resolved infrared difference spectra have been obtained by step-scan Fourier transform infrared spectroscopy. The results showed that the lowest-lying excited state for the complexes were a metal-to-ligand charge transfer (MLCT), a 3(pi)(pi)* states and a mixture of ligand-based and MLCT states.

The Rh-Rh, Rh-C and C triple bond N connections present in the molecular system of the ligand-bridged complex of Rb2(1,3-diisocyanopropane)4(BPh4)2 were studied using time-resolved infrared spectroscopy (TRIR). The excited state displacement of the molecule was monitored in the 2130 to 2200 cm-1 spectral range. The TRIR results accurrately measured the spectra of the three bondings. In addition, the evaluation of the observed displacements identified the behaviour of the complex during excited state chemistry.

Results of the picosecond infrared study of the dynamics of a mixed-valent ruthenium dimer after a metal-metal charge-transfer (MMCT) optical excitation is discussed. The terminal RuC=N stretching frequency is observed and this leads to the detailed study on the formation and decay of the MMCT excited state. The vibrational coupling and energy-transfer dynamics can be understood by observing the energy flow during the de-excitation process. Results show that the back electron transfer from the MMCT state is ultrafast.

In gas turbine combustion the gas dynamic and chemical energy release mechanisms have time scales, so that equilibrium chemistry is inadequate for predicting species formation (emissions). In current practice either equilibrium chemical reactions are coupled with experimentally derived empirical equations, or time-consuming computations are used. Coupling nonequilibrium chemistry, fluid dynamic, and initial and boundary condition equations results in large sets of numerically stiff equations; and their time integration demands enormous computational resources. The response modeling approach has been used successfully for large reaction sets. This paper makes two new contributions. First it shows how pre-integration of the heat release maps eliminates the stiffness of the equations. This is a new modification to the response mapping approach, and it performs satisfactorily for non-diffusion systems. Second the theoretical framework is further extended to predict species formation in cases with diffusion, which is applicable to gas turbine combustion systems and others. The methodology to implement this approach to reacting systems, and to gas turbine combustion, is presented. The benefits over other reaction-mapping techniques are discussed. [ABSTRACT FROM AUTHOR]