Richard Neutze

Potential for biomolecular imaging with femtosecond X-ray pulses

Sample damage by X-rays and other radiation limits the resolution of structural studies on non-repetitive and non-reproducible structures such as individual biomolecules or cells. Cooling can slow sample deterioration, but cannot eliminate damage-induced sample movement during the time needed for conventional measurements. Analyses of the dynamics of damage formation suggest that the conventional damage barrier (about 200 X-ray photons per Å2 with X-rays of 12 keV energy or 1 Å wavelength) may be extended at very high dose rates and very short exposure times. Here we have used computer simulations to investigate the structural information that can be recovered from the scattering of intense femtosecond X-ray pulses by single protein molecules and small assemblies. Estimations of radiation damage as a function of photon energy, pulse length, integrated pulse intensity and sample size show that experiments using very high X-ray dose rates and ultrashort exposures may provide useful structural information before radiation damage destroys the sample. We predict that such ultrashort, high-intensity X-ray pulses from free-electron lasers that are currently under development, in combination with container-free sample handling methods based on spraying techniques, will provide a new approach to structural determinations with X-rays.

Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin

A wide variety of mechanisms are used to generate a proton-motive potential across cell membranes, a function lying at the heart of bioenergetics. Bacteriorhodopsin, the simplest known proton pump, provides a paradigm for understanding this process. Here we report, at 2.1 Å resolution, the structural changes in bacteriorhodopsin immediately preceding the primary proton transfer event in its photocycle. The early structural rearrangements propagate from the proteins core towards the extracellular surface, disrupting the network of hydrogen-bonded water molecules that stabilizes helix C in the ground state. Concomitantly, a bend of this helix enables the negatively charged primary proton acceptor, Asp 85, to approach closer to the positively charged primary proton donor, the Schiff base. The primary proton transfer event would then neutralize these two groups, cancelling their electrostatic attraction and facilitating a relaxation of helix C to a less strained geometry. Reprotonation of the Schiff base by Asp 85 would thereby be impeded, ensuring vectorial proton transport. Structural rearrangements also occur near the proteins surface, aiding proton release to the extracellular medium.

Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle.

Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics.

Observable frequency shifts via spin-rotation coupling

The phase perturbation arising from spin-rotation coupling is developed as a natural extension of the celebrated Sagnac effect. Experimental evidence in support of this phase shift, however, has yet to be realized due to the exceptional sensitivity required. We draw attention to the relevance of a series of experiments establishing that circularly polarized light, upon passing through a rotating half-wave plate, is changed in frequency by twice the rotation rate. These experiments may be interpreted as demonstrating the role of spin-rotation coupling in inducing this frequency shift, thus providing direct empirical verification of the coupling of the photon helicity to rotation. A neutron interferometry experiment is proposed which would be sensitive to an analogous frequency shift for fermions. In this arrangement, polarized neutrons enter an interferometer containing two spin flippers, one of which is rotating while the other is held stationary. An observable beating in the transmitted neutron beam intensity is predicted.

Deconvoluting ultrafast structural dynamics: temporal resolution beyond the pulse length of synchrotron radiation

100 picosecond X-ray snapshots visualizing the structural dynamics of macromolecular systems are now routinely available at synchrotron sources. A wealth of fundamental processes in photochemistry, condensed matter physics and biology, however, occur on considerably faster time scales. Standard experimental protocols at synchrotron sources cannot provide structural information with faster temporal resolution as these are limited by the duration of the electron bunch within the synchrotron ring. By walking the timing of femtosecond laser photolysis through a (much longer) X-ray pulse in steps of a few picoseconds, structural information on ultrafast dynamics may be retrieved from a set of X-ray scattering images, initially through deconvolution and subsequently through refinement. This experimental protocol promises immediate improvements in the temporal resolution available at synchrotron sources, facilitating the study of a number of rapid complex photochemical processes. Combined with techniques which reshape the X-ray probe pulse, the accessible temporal domain could further be extended to near-picosecond resolution.

Ultrafast structural studies on biological molecules by x-rays

Dramatic changes can be expected within the next few years in science that depends on synchrotron radiation today. An analysis of available data on short-pulse, high-intensity x-ray and electron sources indicate possibilities for imminent developments with x-ray free-electron lasers (driven primarily by the high energy physics community), and with table-top femtosecond x-ray sources based on laser-induced plasmas and wakefield acceleration. New sources could generate femtosecond x-ray pulses with as much as 12 orders of magnitude increase in peak brilliance and power over third-generation synchrotron storage rings. Such developments would create revolutionary new research opportunities in condensed matter physics, biology and medicine. Some of the possibilities are discussed here.