A molecular movie at 1.8 A resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds.

The photocycle of the bacterial blue-light photoreceptor, photoactive yellow protein, was stimulated by illumination of single crystals by a 7 ns laser pulse. The molecular events were recorded at high resolution by time-resolved X-ray Laue diffraction as they evolved in real time, from 1 ns to seconds after the laser pulse. The complex structural changes during the photocycle at ambient temperature are displayed in a movie of difference electron density maps relative to the dark state. The step critical to entry into the photocycle is identified as flipping of the carbonyl group of the 4-hydroxycinnamic acid chromophore into an adjacent, hydrophobic environment rather than the concomitant isomerization about the double bond of the chromophore tail. The structural perturbation generated at the chromophore propagates throughout the entire protein as a light-induced "protein quake" with its "epicenter" at the carbonyl moiety of the chromophore.

A mammalian expression vector for expression and purification of secreted proteins for structural studies.

A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined.

New techniques in fast time-resolved structure determination.

New techniques in fast time-resolved X-ray crystallography provide a different approach to understanding the structural basis of protein function. Two biological systems have been studied as part of the refinement of these techniques, and have actually spurred new ideas in time-resolved structural studies. The dissociation of carbon monoxide from carbon-monoxy myoglobin has earlier been investigated over a time range spanning 18 orders of magnitude (femtoseconds to hours) using spectroscopic methods. Rapid time-resolved determination of the entire myoglobin structure made it possible to determine both the position of the CO after photodissociation and the entire globin structure, over a time range from nanoseconds to milliseconds, during which the heme and globin relax and the carbon monoxide rebinds. Photoactive yellow protein, a relative newcomer to biophysical research, has a fully-reversible photocycle containing several spectrally distinct intermediates. Identifying and solving the structures of each intermediate is the initial goal in time-resolved studies on this protein and will contribute to a greater understanding of the biological process of light driven signal transduction.

Extraction of accurate structure-factor amplitudes from Laue data: wavelength normalization with wiggler and undulator X-ray sources.

Wavelength normalization is an essential part of processing of Laue X-ray diffraction data and is critically important for deriving accurate structure-factor amplitudes. The results of wavelength normalization for Laue data obtained in nanosecond time-resolved experiments at the ID09 beamline at the European Synchrotron Radiation Facility, Grenoble, France, are presented. Several wiggler and undulator insertion devices with complex spectra were used. The results show that even in the most challenging cases, such as wiggler/undulator tandems or single-line undulators, accurate wavelength normalization does not require unusually redundant Laue data and can be accomplished using typical Laue data sets. Single-line undulator spectra derived from Laue data compare well with the measured incident X-ray spectra. Successful wavelength normalization of the undulator data was also confirmed by the observed signal in nanosecond time-resolved experiments. Single-line undulators, which are attractive for time-resolved experiments due to their high peak intensity and low polychromatic background, are compared with wigglers, based on data obtained on the same crystal.

Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle.

Photoactive yellow protein (PYP) is a member of the xanthopsin family of eubacterial blue-light photoreceptors. On absorption of light, PYP enters a photocycle that ultimately transduces the energy contained in a light signal into an altered biological response. Nanosecond time-resolved x-ray crystallography was used to determine the structure of the short-lived, red-shifted, intermediate state denoted [pR], which develops within 1 nanosecond after photoelectronic excitation of the chromophore of PYP by absorption of light. The resulting structural model demonstrates that the [pR] state possesses the cis conformation of the 4-hydroxyl cinnamic thioester chromophore, and that the process of trans to cis isomerization is accompanied by the specific formation of new hydrogen bonds that replace those broken upon excitation of the chromophore. Regions of flexibility that compose the chromophore-binding pocket serve to lower the activation energy barrier between the dark state, denoted pG, and [pR], and help initiate entrance into the photocycle. Direct structural evidence is provided for the initial processes of transduction of light energy, which ultimately translate into a physiological signal.

Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration.

BPAG1 is the major antigenic determinant of autoimmune sera of bullous pemphigoid (BP) patients. It is made by stratified squamous epithelia, where it localizes to the inner surface of specialized integrin-mediated adherens junctions (hemidesmosomes). To explore the function of BPAG1 and its relation to BP, we targeted the removal of the BPAG1 gene in mice. Hemidesmosomes are otherwise normal, but they lack the inner plate and have no cytoskeleton attached. Though not affecting cell growth or substratum adhesion, this compromises mechanical integrity and influences migration. Unexpectedly, the mice also develop severe dystonia and sensory nerve degeneration typical of dystonia musculorum (dt/dt) mice. We show that in at least one other strain of dt/dt mice, BPAG1 gene is defective.

Reading attainment in hearing-impaired children: a comparison of higher and lower achievers.

The purpose of the present research is to study hearing-impaired children with higher and lower reading attainment in order to suggest why some of these choldren achieve greater competency than others. Sixteen children, ages 8 to 17, from two oral schools for the hearing-impaired in Britain, were given two tests of reading comprehension. These revealed (1) marked differences in the reading levels of the children, and (2) a relationship between the test scores and what school the children attended. Differences were also found when the children's oral language use and reading comprehension levels were compared. Oral language use was shown to be an important skill related to attainment in reading. Differences in the nature of early language experiences of children in the two schools were said to be partly responsible for the disparity in the children's oral language ability and the differences in their later ability to read. Some insights are offered to explain how one school has fostered the development of good reading skills where other schools have failed to do so.