MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites.

MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt(2+) ions were found to bind at the same place in the structure. The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively.

Two crystal forms of ModE, the molybdate-dependent transcriptional regulator from Escherichia coli.

The molybdenum-responsive ModE regulatory protein from Escherichia coli has been purified and used in crystallization trials. Two crystal forms have been observed. Form I is tetragonal, P41212 (or enantiomorph), with a = b = 72.3, c = 246.2 A and diffracts to medium resolution. Form II is orthorhombic, P21212, with a = 82.8, b = 127.9, c = 64.0 A and diffraction has been observed beyond 2.8 A resolution. Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationship in regulating the uptake of molybdate in bacteria.

The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds.

The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum. We have determined the structure of ModE using multi-wavelength anomalous dispersion. Selenomethionyl and native ModE models are refined to 1. 75 and 2.1 A, respectively and describe the architecture and structural detail of a complete transcriptional regulator. ModE is a homodimer and each subunit comprises N- and C-terminal domains. The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization. The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified. This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies. The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions. This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription. Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation.

Structure determination of a 16.8 kDa copper protein at 2.1 A resolution using anomalous scattering data with direct methods.

The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 A resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f" = 3.9 e-) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr approximately /= 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (approximately 2 A) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f" approximately 4 e-), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f"max++ = 8 e- at the 'white line') and 80 kDa for a Pt derivative (f"max = 19 e- at the 'white line', L3 edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data.

Beamline 14: a new multipole wiggler beamline for protein crystallography on the SRS.

A new multipole wiggler device has been designed for the 2.0 GeV Synchrotron Radiation Source at Daresbury Laboratory in the UK. The nine-pole 2.0 T device will provide radiation for two beamlines dedicated to protein crystallography, one of which will be of high intensity. This article provides details of the design of the two stations and outlines methods being developed to combine dealing with the high heat load from the radiation while allowing both stations to be built as close to the centre of the fan as possible.

PXGEN: a General-Purpose Graphical User Interface for Protein Crystallography Experimental Control and Data Acquisition.

PXGEN is a general-purpose graphical user interface for experimental set-up and control of protein crystallography data collection. PXGEN is not linked intrinsically to any software package or proprietary hardware and should be transportable to other experimental facilities. The experimental techniques supported are single-wavelength data collection and multiwavelength anomalous dispersion. The graphical user interface runs on a UNIX-based workstation exploiting the host's power to manage multiple programs. PXGEN provides a mechanism for making data collection much easier and less error-prone. The design and implementation of PXGEN are described, which is now installed on protein crystallography beamlines 9.5 and 7.2 of the Synchrotron Radiation Source at Daresbury Laboratory.

Preliminary crystallographic studies of dimethylsulfoxide reductase from Rhodobacter capsulatus.

Dimethylsulfoxide reductase from the photosynthetic bacterium Rhodobacter capsulatus has been crystallized in two similar forms which are suitable for X-ray structure determination. Both crystals forms belong to space group P4(1)22 or P4(3)22, with cell dimensions a = b = 80.81, c = 229.75 A (type I crystals) or a = b = 89.30, c = 230.05 A (type II crystals) and one molecule in the asymmetric unit. Diffraction has been observed to at least 2.0 A in type I crystals and to 2.6 A in type II crystals. Dimethylsulfoxide reductase from Rhodobacter is the simplest molybdenum oxotransferase known and this makes it an ideal model to study the structure and function of this class of enzymes.

Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

Towards time-resolved diffraction studies with glycogen phosphorylase.

Laue diffraction with high intensity, broad-spectrum synchrotron radiation sources allows three-dimensional data sets on protein crystals to be recorded in seconds or milliseconds and opens the way for time-resolved studies on dynamic events in crystals. This chapter briefly reviews the field and describes progress towards time-resolved studies with glycogen phosphorylase. Methods for the synchronization of the start of reaction with the start of data collection have been developed for the phosphorolytic reaction of glycogen phosphorylase. The compound 3,5-dinitrophenylphosphate is photolabile, yielding Pi and the by-product, 3,5-dinitrophenol, which is non-reactive with the enzyme. Spectroscopic studies show that the compound has good quantum yield and that photolysis is rapid (greater than 1000 s-1). Release of the dinitrophenylate anion, following a pulse of light from a xenon flash lamp, has been monitored with a diode array spectrophotometer specially adapted for measurements on crystals. In a laboratory X-ray experiment with crystals of glycogen phosphorylase b, release of Pi and formation of the enzyme-product complex have been demonstrated. The way is now open for Laue diffraction studies on the catalytic reaction in the crystal.

Thyroid dysfunction in Down's syndrome.

One hundred and sixteen children with Down's syndrome, living in the community, were examined for clinical or laboratory evidence of thyroid dysfunction. Three were hypothyroid and one was hyperthyroid. Twenty eight (29%) had thyroid autoantibodies. Autoimmune conditions were present in first or second degree relatives of 35 (30%) of the children, and in 17 (15%) this was a thyroid disorder. The families of normal control children also showed a 30% incidence of overt autoimmune conditions, and 19 (16%) families showed overt thyroid disease.

Carrier detection in the testicular feminisation syndrome: deficient 5 alpha-dihydrotestosterone binding in cultured skin fibroblasts from the mothers of patients with complete androgen insensitivity.

Specific binding of 5 alpha-dihydrotestosterone (androgen receptor activity) could not be detected in cultured genital skin fibroblasts (GSF) from two patients with complete androgen insensitivity (CAI). In GSF from the mother of one patient, androgen receptor activity (8.5 fmol/mg cell protein) was reduced in comparison with controls (34.0 +/- 10.1 (SD) fmol/mg protein n = 15). These results favour X linked inheritance of CAI and X inactivation at the androgen receptor locus. Androgen receptors were not detected in GSF of the second mother. It appears that female carriers of CAI could be detected by decreased 5 alpha-dihydrotestosterone binding in GSF. Androgen receptor activity was also undetectable in non-genital skin fibroblasts (NGSF) from the second mother and two further CAI patients. However, in 1 in 10 control NGSF lines androgen receptor activity was at the lower limit of assay sensitivity (1 to 2 fmol/mg protein) demonstrating that NGSF may not be reliable for family studies of androgen receptor deficiency.

Turner's syndrome associated with growth hormone deficiency.

11 out of the 13 children with Turner's syndrome currently attending our endocrine clinic were investigated for possible growth hormone deficiency. The parents of two of the 13 children refused permission for these studies. One child had inadequate hypoglycaemia and the test was not repeated. Six of the ten children with adequate hypoglycaemia had an adequate growth hormone response to hypoglycaemia, while 4 children did not. This contradicts several previous studies on children with Turner's syndrome, which have reported normal growth hormone responses to provocative tests. In the normal population approximately one in 15 has an inadequate growth hormone response to hypoglycaemia.