Biomedical subjects

Keith Moffat

Publications and source records attributed to Keith Moffat.

23 records · Page 2Linked to original sources

PubMed2003

The frontiers of time-resolved macromolecular crystallography: movies and chirped X-ray pulses.

Three important frontiers of ultrafast time-resolved macromolecular crystallography are presented: extension of this technique to other biological systems; further developments in the elucidation of mechanism through the analysis of time-dependent movies to extract the underlying, time-independent, intermediate structures; and enhanced time resolution. The last is intimately linked with the nature of the pump-probe experiment itself, with the sources of random and, particularly, systematic experimental error, and with the factors that contribute to overall time resolution. All experiments to date have utilized the unchirped X-ray pulses that are emitted by synchrotron sources. Chirped pulses offer certain advantages for ultrafast X-ray experiments such as those based on Laue diffraction. An energy-chirped pulse maps photon energy into time; a Laue diffraction experiment maps photon energy into detector space. Hence, a Laue experiment with an energy-chirped pulse maps time into space. The proposed sub-picosecond photon source could provide an excellent source of intense, chirped hard X-rays for such experiments.

Bacterial Proteins↗

PubMed2002

Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K.

X-ray radiation damage of lysozyme single crystals by an intense monochromatic beam from the Advanced Photon Source is studied at cryogenic temperatures between 40 K and 150 K. The results confirm that primary radiation damage is both linearly dependent on the X-ray dose and independent of temperature. The upper limit for the primary radiation damage observed in our previous study [Teng & Moffat (2000), J. Synchrotron Rad. 7, 313-317] holds over the wider temperature range of this study. The X-ray diffraction quality of the data acquired at 40 K is superior to those at 100 K, apparently due to temperature dependence of secondary and tertiary radiation damage and to reduced thermal motion.

Animals↗

PubMed2002

Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch.

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain. The structure reveals a unique photochemical switch in the flavin binding pocket in which the absorption of light drives the formation of a reversible covalent bond between a highly conserved Cys residue and the flavin cofactor. This provides a molecular picture of a cysteinyl-flavin covalent adduct, the presumed signaling species that leads to phototropin kinase activation and subsequent signal transduction. We identify closely related LOV domains in two eubacterial proteins that suggests the light-induced conformational change evident in this structure is an ancient biomolecular response to light, arising before the appearance of plants.

Amino Acid Sequence↗

PubMedDate not supplied

Structural heterogeneity of cryotrapped intermediates in the bacterial blue light photoreceptor, photoactive yellow protein.

We investigate by X-ray crystallographic techniques the cryotrapped states that accumulate on controlled illumination of the blue light photoreceptor, photoactive yellow protein (PYP), at 110 K in both the wild-type species and its E46Q mutant. These states are related to those that occur during the chromophore isomerization process in the PYP photocycle at room temperature. The structures present in such states were determined at high resolution, 0.95-1.05A. In both wild type and mutant PYP, the cryotrapped state is not composed of a single, quasitransition state structure but rather of a heterogeneous mixture of three species in addition to the ground state structure. We identify and refine these three photoactivated species under the assumption that the structural changes are limited to simple isomerization events of the chromophore that otherwise retains chemical bonding similar to that in the ground state. The refined chromophore models are essentially identical in the wild type and the E46Q mutant, which implies that the early stages of their photocycle mechanisms are the same.

Bacterial Proteins↗

PubMedDate not supplied

Purification and initial characterization of a putative blue light-regulated phosphodiesterase from Escherichia coli.

The Escherichia coli protein YcgF contains a photosensory flavin adenine dinucleotide (FAD)-binding BLUF domain covalently linked to an EAL domain, which is predicted to have cyclic-di-guanosine monophosphate (GMP) phosphodiesterase activity. We have cloned, overexpressed and purified this protein, which we refer to as blue light-regulated phosphodiesterase (Blrp) for its putative activity. Blrp undergoes a reversible photocycle after exposure to light in which the spectrum of its photostationary state and kinetics of recovery of the dark state are similar to those of the isolated BLUF domain of the AppA protein. Unlike the AppA BLUF domain, the chromophore environment in the context of full-length Blrp is asymmetric, and the protein does not undergo any detectable global changes on exposure to blue light. When overexpressed in E. coli, Blrp copurifies with certain proteins which suggests that it plays a protective role in response to oxidative stress. Predicted proteins from Klebsiella pneumoniae and from a bacterium in the Sargasso Sea are similar to E. coli Blrp in both their BLUF and EAL domains, which suggests that blue light sensing in these bacteria may follow similar pathways.

Amino Acid Sequence↗