Neutron structure of monoclinic lysozyme crystals produced in microgravity

Crystals of the monoclinic form of lysozyme (space group P2(1) with a = 28.00 Angstrom, b = 62.88 Angstrom, c = 60.30 Angstrom, beta = 90.68 degrees) were grown under microgravity conditions on the Mir station and brought back to earth on the US Space Shuttle. Counter-diffusion methods developed specifically for application in microgravity have been utilized to produce several examples of macroscopic crystals. Large crystals are of great importance for neutron diffraction studies of bio-macromolecules, which can reveal key details of the hydrogen atom structure of biological molecules at medium resolution. The structure of the monoclinic crystal form described here has been determined to 2.1 Angstrom by neutron diffraction and contains two molecules in the asymmetric unit. Details of the structure and refinement are presented. (C) 2001 Elsevier Science B.V. All rights reserved

Directed microbial biosynthesis of deuterated biosurfactants and potential future application to other bioactive molecules

Deuterated rhamnolipids were produced using strain AD7 of Pseudomonas aeruginosa, which was progressively adapted to increasing levels of deuterium in D(2)O and carbon substrates. Fourteen different deuterated rhamnolipid structures, including structural isomers, were produced which is similar to normal protonated structures. There were two main products monorhamnolipid Rha-C(10)-C(10) and dirhamnolipid Rha(2)-C(10)-C(10). The levels of deuteration varied from 16% with 25% D(2)O + h-glycerol to 90% with 100% D(2)O + d-glycerol. When d-tetradecane was used with H(2)O, virtually all the deuterium appeared in the lipid chains while using h-tetradecane + D(2)O led to the majority of deuterium in the sugars. The adaptation to growth in deuterium appeared to be metabolic since no genetic changes could be found in the key rhamnolipid biosynthetic genes, the rhamnosyl transferases RhlB and RhlC. Deuterated sophorolipids were similarly produced using Candida bombicola and Candida apicola although in this case, no adaptation process was necessary. Up to 40 different sophorolipids were produced by these yeasts. However, unlike the rhamnolipids, use of D(2)O did not lead to any deuteration of the lipid chains, but direct incorporation into the lipid was achieved using d-isostearic acid. The results from these experiments show the feasibility of producing deuterated bioactive compounds from microorganisms coupled with the possibility of manipulating the pattern of labelling through judicious use of different deuterated substrates

X-ray structure of a decameric cyclophilin-cyclosporin crystal complex

Human cyclophilin A (CypA), a ubiquitous intracellular protein of 165 amino acids, is the major receptor for the cyclic undecapeptide immunosuppressant drug cyclosporin A (CsA), which prevents allograft rejection after transplant surgery and is efficacious in the field of autoimmune diseases. CsA prevents T-cell proliferation by blocking the calcium-activated pathway leading to interleukin-2 transcription. Besides their ability to bind CsA, the cyclophilin isoforms also have peptidyl-prolyl isomerase activity and enhance the rate of protein folding. The macrolide FK506 acts similarly to CsA and its cognate receptor FKBP also has peptidyl-prolyl isomerase activity. Inhibition of this enzymatic activity alone is not sufficient to achieve immunosuppression. A direct molecular interaction between the drug-immunophilin complex (CsA-CypA, or FK506-FKBP) and the phosphatase calcineurin, is responsible for modulating the T-cell receptor signal transduction pathway. Here we describe the crystal structure of a decameric CypA-CsA complex. The crystallographic asymmetric unit is composed of a pentamer of 1:1 cyclophilin-cyclosporin complexes of rather exact non-crystallographic fivefold symmetry. The 2.8 A electron density map is of high quality. The five independent cyclosporin molecules are clearly identifiable, providing an unambiguous picture of the detailed interactions between a peptide drug and its receptor. It broadly confirms the results of previous NMR, X-ray and modelling studies, but provides further important structural details which will be of use in the design of drugs that are analogues of CsA