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The dynamics of ligand escape from carboxymyoglobin are studied via Q-band transient absorption and diffractive optics-based four-wave mixing. The latter approach provides an interferometric method for following protein motions and energetics and allows unambiguous assignment of different signal components to specific dynamical processes, a problem that has hindered all photothermal, photoacoustic, and grating methods in the past. In particular, the real part of the four-wave mixing signal is isolated, and it is demonstrated that the excited-state population contribution to the signal can be identified and removed by tuning the probe to wavelengths where this contribution vanishes ("zero-crossings"). At these probe wavelengths, changes in the real part of the index of refraction are small compared to those of the imaginary part, making the heterodyne measurement sensitive to errors in setting the phase of the reference field. We solve this problem with a new balanced detection method that isolates the real part of the signal and is extremely robust against phase errors. The location of the zero-crossings in the population contribution to the index of refraction is found to be complicated by the presence of spectral shifts in the transient absorption that can be characterized and removed with a time-dependent Kramers-Kronig analysis. The spectral shifts are most apparent near the isosbestic points and show similar dynamics to the four-wave mixing signal, suggesting that both are sensitive to the same dynamical processes. These dynamics were observed previously by using four-wave mixing with an off-resonant probe and were assigned to CO migration out of the protein via a number of discrete channels. It appears that both the photoinduced protein conformational relaxation and CO migration through the protein contribute to the transient absorption signal, making the two effects difficult to separate. The direct coupling of the protein motions to the index of refraction changes in the four-wave mixing experiments aids in distinguishing the two processes.

The dynamics of carboxy-myoglobin (MbCO) in water are studied from photodissociation of the ligand to bimolecular recombination over 12 decades in time to fully characterize the protein functions. Heterodyne-detected transient grating spectroscopy is used to resolve the dynamics of the protein from 10 ns to a few ms and provides a direct observation of the ligand escape. The process is well described by a bi-exponential function with decay rates of 50 and 725 ns at 20°C, suggesting that ligand escape occurs via a well defined pathway. Transient absorption in the Q-band (550–630 nm) also reveals the sensitivity of the electronic transition to the ligand motions. The dynamic range is extended by 10 6 through femtosecond coherence spectroscopy with 7 fs pulses to enable the observation of vibrational modes of energies >1500 cm −1 . The power spectrum is calculated by singular value decomposition and vibrational modes involved in the photodissociation are directly observed. The picture that is emerging is that proteins couple solid-like domains to fluid regions to facilitate functions and transport of ligands in and out of the protein to the active site.

Recent advances in transient grating spectroscopy are described in relation to extracting photoacoustic parameters. The time resolution for measurement of dynamically driven acoustics has been extended to the picosecond level. This improvement was achieved by adopting counter-propagating beam geometries and resolving the acoustic phase shift in analogy to phase modulation spectroscopy. At the other extreme, the dynamic range of this technique has been extended to milliseconds in order to follow dynamical processes central to biological functions. In addition, diffractive optics were used for generating the necessary excitation and probe beam geometries. A novel optical setup was developed which permits both the rapid exploration of fringe spacing dependencies in separating thermal from nonthermal contributions to the observed signal as well as heterodyne detection without active feedback. The latter capability significantly increases the signal-to-noise ratio and permits separation of the real and imaginary components from the nonlinear four-wave-mixing signal. Applications of these new methods are demonstrated by following the functionally relevant structural relaxation processes of heme proteins over 10 decades in time.

The potential container penetrating capabilities of THz radiation leads to possible applications for container penetrating sensors for biological hazards. Such an approach requires the presence of distinct THz frequency resonances in the target compounds coupled with sufficiently transparent container materials to allow through container sensing. The results of a THz spectroscopic survey of container and clothing materials are presented along with spectra of materials that were chosen as simulants and markers for illicit biological substances. The spectroscopic data presented show at least partial transparency for materials commonly used for clothing and packaging. We also measure distinct spectral signatures in dipicolinic acid, calcium dipicolinate, peptidoglycan, and 2,6-diaminopimelic acid, biologically significant molecules that are indicative of hazardous spore forming bacteria. These spectra differ significantly from those of the container materials to provide a potential contrast mechanism which could be used for identification.

This paper presents results from a continuing investigation of mixing flowfields and optical gain profiles in HF chemical laser systems by infrared hyperspectral imaging. Chemiluminescent F + H2 reacting flowfields and chemical laser output beams are imaged at a series of wavelengths, 2.6 to 2.9 microns, by a low-order, spectrally scanning Fabry-Perot interferometer mated to an infrared camera. The resulting hyperspectral data cubes define the spectral and spatial distributions of the emission. High-resolution images can be processed to determine spatial distributions of the excited state concentrations of the product HF(v,J), as well as spatial distributions of gain on specific laser transitions. This paper describes the instrumentation and its application to detailed observations of reactant mixing and energy transfer in the product HF(v,J), using a low-pressure laboratory reactor developed at Physical Sciences, Inc. (PSI). The measurements confirm that the PSI reactor generates inverted populations of HF(v,J).

Summary form only given. Biological systems constantly transduce various forms of chemical energy into functions in which the inherent response of the system operates at the edge of stability. Excursions from the stability region lead to denaturation; whereas small fluctuations about the stability point lead to highly correlated responses that behave in a deterministic fashion with respect to the function of the system. Exactly how is the bond energy directed in such a complex system and how has the system evolved to minimize entropic losses in conversion efficiency? We have used the oxygen binding heme proteins as model systems for studying the coupling of reaction forces to functionally relevant motions; i.e., structural transitions important to the self regulation of oxygen binding and transport. Since the forces involved become spatially distributed over an enormous number of degrees of freedom, the net relative motions can be exceedingly small (

Summary form only received as follows: The development of cross-linked Hb artificial blood substitutes is an area of much growth and attention. Where donated blood can be stored for up to three weeks, artificial blood can be stored indefinitely. The problem of disease transmission is negligible. Moreover, these blood substitutes can act as drug-delivery agents as well as having the capacity to deliver oxygen to cancerous sites and aid in photodynamic therapy techniques. In previous work the authors have characterized Hb and Mb using time-resolved phase grating spectroscopy. Recently they have increased the sensitivity of their experiment by implementing heterodyne detection using a diffractive optic. This has allowed them to detect the extremely small changes in structure of these blood substitutes and aid-in their development and understanding. The advent of these artificial blood substitutes also opens up a new venue by which to study energy transduction in biomolecules. This puts the authors in a unique position to test the predictions of the Monod two-state model. By varying the sites of cross-linking they have tested the Monod two-state model for Hb. New results characterizing these cross-linked Hb and probing the validity of the Monod two-state model are shown.

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ± 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

Summary form only given. The relationship between molecular structure and function is of fundamental importance for understanding biological systems. The heme proteins hemoglobin and myoglobin provide ideal model systems for investigating this relationship because their structure and function are well characterized. In addition, they are amenable to optical probes, allowing their functional processes to be initiated by photodissociation. Previous studies on the femtosecond timescale have characterized the dynamics of myoglobin from femtoseconds to nanoseconds. The current work extends these studies to the millisecond regime to capture the full range of functionally relevant motions. These motions are often small and require a highly sensitive spectroscopy for their study. Diffractive optics-based four-wave mixing provides the sensitivity needed to observe changes in radius of