J.P. Ogilvie

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.

Observation of the cascaded atomic-to-global length scales driving protein motion

Model studies of the ligand photodissociation process of carboxymyoglobin have been conducted by using amplified few-cycle laser pulses short enough in duration (<10 fs) to capture the phase of the induced nuclear motions. The reaction-driven modes are observed directly in real time and depict the pathway by which energy liberated in the localized reaction site is efficiently channeled to functionally relevant mesoscale motions of the protein.

Myoglobin dynamics: Evidence for a hybrid solid/fluid state of matter

Abstract 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 10xa0ns 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 725xa0ns at 20°C, suggesting that ligand escape occurs via a well defined pathway. Transient absorption in the Q-band (550–630xa0nm) 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 7xa0fs pulses to enable the observation of vibrational modes of energies >1500xa0cm −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.

Chapter 77 - Reaction driven modes in carboxymyoglobin: pathway of force transduction for functionally relevant protein motions

Heme proteins form the cornerstone for our understanding of the general phenomena of molecular cooperativity; where the binding affinity of diatomic ligands in hemoglobin and associated changes in quaternary structure with ligation state is the best-characterized example. At the heart of this problem is an understanding how the initially localized reaction forces couple to the global protein coordinate to execute these functionally relevant motions. The energetics for this process are derived from one or a few chemical bonds and at the instant the bond is formed or broken, the motions are necessarily localized over atomic length scales at the active site and subject to quantum effects. From a practical standpoint, the heme proteins provide ideal model systems for studying the relationship between protein structure and function since they are well-characterized and their ligands can be photo-dissociated with unit quantum efficiency on the truly femtosecond timescale. This chapter discusses the results of ultrafast pump-probe spectroscopy of carboxymyoglobin (MbCO) to determine the initial structure changes of the ligand dissociation process using pulses of less than 10 fs in the 500-600 nm spectral regions.

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.