We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 1010 clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.

A sample environment for mounting crystallization trays has been developed on the microfocus beamline I24 at Diamond Light Source. The technical developments and several case studies are described.

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4 -33 times higher specificity for DSHA (k cat /K m ‫؍‬ 3.3 ؎ 0.3 ؋ 10 4 M ؊1 s ؊1 ) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K d of 51 ؎ 2 M. The S114A⅐DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8 -1.9 Å indicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the ␣-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å , supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.Mycobacterium tuberculosis is the leading cause of bacterial mortality, causing an estimated 2 million deaths/year (1). The mechanisms underlying the remarkable ability of this pathogen to survive for long periods of time within the host are poorly understood (2). Although it was well known that saprophytic mycobacteria could metabolize cholesterol (3), it was only recently demonstrated that pathogenic strains can also utilize this nutrient as a growth substrate (4, 5). Interestingly, cholesterol has been found in high concentrations within caseating granulomas in both humans and mice (6, 7), and bacteria have been observed congregating around cholesterol foci (7). Highlighting the importance of cholesterol in bacterial pathogenesis, the deletion of genes involved in cholesterol metabolism reduces the virulence of M. tuberculosis (5,8). Therefore, further knowledge of cholesterol metabolism in M. tuberculosis is crucial to our understanding of bacterial virulence.M. tuberculosis catabolizes cholesterol using a metabolic pathway similar to that identified in Rhodococcus jostii RHA1 (4, 9). In this pathway, the aerobic degradation of the fourringed steroid nucleus occurs through the opening of ring B, aromatization of ring A, and hydroxyla...

An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.

A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector.

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes.In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxidetoform the catalytic intermediate compound I(Fe IV = Oa nd ap orphyrin cation radical). Using serial femtosecond X-rayc rystallography (SFX), we have determined the pristine structures of the Fe III and Fe IV =Oredox states of aB-type DyP.These structures reveal aw ater-free distal heme site that, together with the presence of an asparagine,imply the use of the distal arginine as ac atalytic base.Acombination of mutagenesis and kinetic studies corroborate such ar ole.O ur SFX approach thus provides unique insight into howt he distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxideh eterolysis.

Some ideas and methods on how to produce high-quality samples for successful serial crystallography experiments are presented. The methods here described are aimed at experimenters trying to convert their vapour diffusion crystallization conditions into large-scale batch micro-crystallization.

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