Time-resolved crystallography and protein design: signalling photoreceptors and optogenetics

Time-resolved X-ray crystallography and solution scattering have been successfully conducted on proteins on time-scales down to around 100 ps, set by the duration of the hard X-ray pulses emitted by synchrotron sources. The advent of hard X-ray free-electron lasers (FELs), which emit extremely intense, very brief, coherent X-ray pulses, opens the exciting possibility of time-resolved experiments with femtosecond time resolution on macromolecular structure, in both single crystals and solution. The X-ray pulses emitted by an FEL differ greatly in many properties from those emitted by a synchrotron, in ways that at first glance make time-resolved measurements of X-ray scattering with the required accuracy extremely challenging. This opens up several questions which I consider in this brief overview. Are there likely to be chemically and biologically interesting structural changes to be revealed on the femtosecond time-scale? How shall time-resolved experiments best be designed and conducted to exploit the properties of FELs and overcome challenges that they pose? To date, fast time-resolved reactions have been initiated by a brief laser pulse, which obviously requires that the system under study be light-sensitive. Although this is true for proteins of the visual system and for signalling photoreceptors, it is not naturally the case for most interesting biological systems. To generate more biological targets for time-resolved study, can this limitation be overcome by optogenetic, chemical or other means?     

A system for accurate immunolocalization of Tamm-Horsfall protein in renal biopsies

Summary The distribution of Tamm-Horsfall protein (THP) was studied in the human kidney using formalin-fixed, paraffin-embedded sections with a monoclonal antibody specific for human THP applied in conjunction with a modified dinitrophenyl hapten sandwich staining (DHSS) procedure.The method was found to be highly sensitive producing very strong specific staining at antibody dilations up to 1 in 64 000. Counterstaining with Haematoxylin and Eosin was possible without significant masking of the specific staining. This provided excellent structural definition of the background tissue, which proved especially important in the study of THP localization in randomly oriented biopsy material. THP was found in all segments of the thick ascending limbs of loops of Henle, most segments of distal convoluted tubules and occasionally in distended collecting ducts and in the glomerular capsular space. Maculae densae did not contain THP.The combination of the modified DHSS procedure and the human THP specific antibody represents a highly sensitive and reliable method for specific staining of the THP in kidney sections.     

W. Montague Cobb (1904–1990): Physical Anthropologist, Anatomist, and Activist

William Montague Cobb's life and work reflect a profound integration of art, literature, social activism, and science. This article presents some of the highlights of his academic development and professional contributions. We have considered his early academic development within the contexts of the formative years of American physical anthropology, Howard University Medical School, and the social issues in American society that influenced Cobb. His approaches to teaching, anatomical and anthropological research, and medicine are unique, and yet are closely reasoned and creative reflections of the major currents of academe and the broader society with which he dealt. Imbued with a sense of social responsibility, Cobb's applied anthropology involved the accumulation of extensive data on the one hand, and the formation of organizations for social activism on the other. It was directed toward solving problems of health care and racism. His work thereby served to balance the widespread distortion and neglect of medical and racial problems facing A fro-America between 1930 and the present day. He was also a principal builder of black medical and scientific institutions, and he preserved the record of his coworkers' contributions through his many biographies. This work represents no more than a sketch of his rich and prolific career (during which he produced more than 1,100 publications); the emphasis of this biographical study has been to ascertain the circumstances and attitudes that helped mold the first Afro-American Ph.D. in physical anthropology.     

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.