Gami Dadusc

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

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.

Diffractive optics-based heterodyne detected four-wave mixing studies of protein dynamics: insights into ligand escape and cooperativity in heme proteins

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 <0.001 /spl Aring/. The use of diffractive optics facilitates the separation of Real and Imaginary parts of the /spl chi//sup 3/ signal by providing the required beam geometry for mixing the signal with a reference beam. In addition it offers passive phase-stabilization. A novel detection method that exploits the symmetry of the four-wave mixing experiment has been implemented to provide automatic isolation of the Real part of the signal. This simplifies the interpretation of the data by obviating the need to identify the Imaginary part of the signal. Further improvement in the signal-to-noise is an added benefit of this method.

Diffractive optics-based nonlinear spectroscopy: application to the study of deterministic protein motion

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 (<.1 /spl Aring/). A very sensitive method is needed to detect these motions and the time resolution must be sufficient to follow from the very first events of bond breaking to full relaxation. The use of diffractive optics for the implementation of heterodyne detected grating spectroscopy has recently been demonstrated. The diffractive optic also generates tilted phase fronts to provide true femtosecond time resolution in noncollinear geometries. This approach has sufficient time resolution and sensitivity to follow the mass displacement, as connected through changes in the material index of refraction, to address this issue.

Characterizing artificial blood substitutes and testing the Monod two-state model using heterodyned phase-grating spectroscopy

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.