Astbury Centre for Structural Molecular Biology

cover picture

Annual Report 2001
© University of Leeds 2002

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Front cover illustration:
The picture shows the magnitude of picosecond internal motions in the backbone of the verotoxin B-subunit (VTB) as measured by NMR spectroscopy using a 1H-15N heteronuclear nOe experiment at a magnetic field strength of 11.4T. The width of the tube is approximately proportional to the degree of motions at this timescale and traces the backbone of a monomer from the X-Ray crystal structure [1BOV]. Secondary structural elements are colour-coded: loops are grey, a -helices are red and b -sheets are blue.

Verotoxin is a AB5 bacterial toxin from E. coli O157:H7 and the VTB homopentamer is responsible for toxin attachment to the target cell. This molecular recognition event results in endocytosis of the A subunit into the cytoplasm of kidney cells resulting in renal failure due N-ribosylation of the ribosome. Each monomer of VTB consists of 69 residues making up of two b -sheets and an a -helix. The data shown is for VTB complexed with S101, an analogue of its natural ligand Gb3 (globotriaosylceramide). This ligand binds to the loop in the bottom right hand corner. The data shown along with other heteronuclear relaxation data are being used to determine conformational entropies of ligand binding on a per residue basis.

Acknowledgement

The Astbury Centre for Structural Molecular Biology thanks its many sponsors for support of the work, its members for writing these reports and Jenny Gilmartin for her hard work in collating and type-setting this report.

 

Mission Statement

The Astbury Centre for Structural Molecular Biology will promote interdisciplinary research of the highest standard on the structure and function of biological molecules, biomolecular assemblies and complexes using physico-chemical, molecular biological and computational approaches.

 

Introduction

It is a great pleasure as the newly appointed Director of the Astbury Centre for Structural Molecular Biology (ACSMB) to write the introduction to this, our second Annual Report. The ACSMB is an interdisciplinary research centre of the University of Leeds. It was founded in 1999 to carry out research in all aspects of structural molecular biology. ACSMB brings together over fifty academic staff from the faculties of Biological Sciences, Chemistry, Physics and Medicine to examine the molecular mechanisms of life. It is named in honour of W.T. (Bill) Astbury, a biophysicist who laid many of the foundations of the field during a long research career at the University of Leeds (1928-1961). Astbury originally identified the two major recurring patterns of protein structure (a and b ), took the first X-ray fibre diffraction pictures of DNA (in 1938) and is widely credited with the definition of the field of molecular biology.

The ACSMB today continues this tradition and focuses on the determination of the atomic structures of biological macromolecules using all of the current major techniques (X-ray diffraction, nuclear magnetic resonance spectroscopy and electron microscopy). ACSMB also specialises in new biophysical techniques such as measuring the mechanical folding properties of individual protein and nucleic acid molecules using atomic force microscopy. These studies are combined with analyses of biological function with the ultimate aim of understanding the molecular basis of life itself. During the last year, ACSMB received a major infrastructure-funding boost (~ 7.5M) from The Wellcome Trust, allowing us to further upgrade our facilities by forming The Wellcome Trust Centre for Biomolecular Interactions. I look forward to seeing the fruits of these new activities in next year's Annual Report.

Structural molecular biology plays a pivotal role in modern biology, both in the fundamental understanding of living things and in the design of new treatments for disease. It is currently undergoing a revolution in both scale and scope. The publication of the human genome sequence holds out the prospect of using structural molecular biology techniques to determine the exact molecular structure of every protein in our bodies. New drugs are frequently designed by analysing the molecular structures of the biological molecules they target, so this promises to generate dramatic advances in healthcare. It is notable that the original observation by Astbury of transitions in fibrous proteins between a and b forms remains relevant today, since devastating amyloid diseases such as Alzheimer's, BSE and CJD are linked to such transitions in brain proteins.

The pages that follow describe some of the highlights of our work over the last year. These reports have largely been written by our younger researchers. Their tremendous enthusiasm for this kind of interdisciplinary work augurs well for our future. I was particularly struck by the breadth of activity in the Centre ranging from the sophisticated applications of synthetic organic chemistry by the Nelson and Warriner groups to the developments in single molecule biophysics to probe early events in protein folding by Smith and Radford. In between these extremes you will find groundbreaking activity in many traditional areas for structural biology. ACSMB has always been outward looking and this tradition continues with the many external collaborations acknowledged in these pages, from both within the UK and beyond. We would welcome discussions with anyone wishing to collaborate or simply to make use of our facilities, the details of which can be found on our web page (http://www.astbury.leeds.ac.uk/).

These brief summaries, however, only scratch the surface of the work of the Centre. I hope you enjoy reading them, and if you wish to learn more please visit our website or contact the Director. This annual report is also available as a 20MB PDF document that can be downloaded.

Peter G. Stockley
Director, Astbury Centre for Structural Molecular Biology
Leeds, March 2002

 

INDEX

Ashcroft - Mass spectrometry facility

Baldwin - Molecular studies on the bacterial nucleoside transporter NupC

Baldwin - Molecular studies on the exocyst complex

Baron - Analytical centrifuge facility

Berry - NMR investigation of the dynamics of the 78kDa Class II fructose-6,6-biphosphate aldolase

Berry - Protein engineering of aldolase: directed evolution and mechanistic enzymology

Berry - Sialic acid-synthesising enzymes: Directed evolution and mechanastic enzymology

Donnelly - Structure/function studies of the first extracellular loop of the glucagon-like peptide-1 receptor

Findlay - Peripherin and retinopathies

Harris - Structural and functional studies of Hepatitis C virus non-structural proteins

Harris - Strcutural and functional stidues on the HIV-1 Nef protein

Henderson - Amplified expression, purification and characterisation of antibiotic resistance transport proteins

Homans - Derivation of per-residue thermodynamic parameters for ligand-protein interactions

Homans - Disecting the ligand binding energy for the E. coli heat-labine toxin

Homans - Efficient resonance assignment and global fold determination of backbone labelled proteins

Homans - Molecular dynamics in mouse urinary protein by NMR methods

Homans - The NMR facility

Homans - NMR studies on the dymanics and structure of Verotoxin-1 B subunits binding to its functional plasma membrane receptor, globotriaosylceramide, Gb3

Homans - The NMR solution structure of the VMA-7 subunit of the vacuolar H+-ATPase from Saccharomyces cerevisiae

Jackson - A new method for comparing ligand binding sites in biomolecules

Jackson - Molecular modelling of SH2 domain-peptide interactions

Jackson - Q-fit: A method for docking molecular fragments by sampling low energy conformational space

Jager - Complexes of Hepatitis C virus RNA polymerase: insights into nucleotide import and de novo priming

Keen - Proteomics and allied technologies

McDowall - An essential activity at the centre of a macromolecular machine

McDowall - Correlating molecular structure and cellular function using microarrays

McDowall - Using reverse genetics & genome data to identify transcriptional regulators

McPherson - Galactose oxidase precursor processing studies

McPherson - Investigating E. coli copper amine oxidase by mutational and inhibitor studies

Nelson - Towards complete stereocontrol: Synthesis of seven of a possible eight C-substituted monosaccharide mimetics

Phillips - Crystal structures of four chemically modified RNA translational operators complexed with bacteriophage MS2 coat protein

Phillips - Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

Phillips - Structural studies of copper amine oxidase

Phillips - Structural studies of processing in galactose oxidase

Ponnambalam - Regulation of membrane protein localisation in eukaryote systems

Radford - Biophysical studies of the beta2-microglobulin amyloid formation

Radford - Folding and misfolding of the four-helix proteins Im7 and Im9

Radford - Probing the determinants of the mechanical resistance in proteins

Rowlands - Receptor mediated attachment of picornaviruses to liposomes and membrane translocation of genome RNA

Rowlands - Structural RNA elements involved in internal initiation of translation

Smith - Application of scanning near-field Raman spectroscopy to biological samples

Smith - Atomic force microscopy of lipid bilayers and membrane bound proteins

Smith - Early stages of protein folding studied by sub-millisecond mixing

Smith - Investigating early events in protein folding by laser induced temperature jump

Smith - Single molecule spectroscopy to probe folding of individual proteins

Smith - Untraviolet resonance Raman spectroscopy in protein folding investigated by microsecond mixing

Smith - Ultraviolet resonance Raman study of streptavidin binding of biotin and 2-iminobiotin

Stockley - Analysing transcription of chromatin templates using surfaceplasmon resonance (SPR)

Stockley - Robotic selection of RNA aptamers against amyloid proteins and peptides

Stonehouse - Molecular interactions in the assembly of bacteriophage O 29

Thomas - Rolling circle replication

Thomson - Beta2-microglobulin amyloid fibril formation in vitro

Thomson - Mechanism of action of DNA gyrase

Thomson - The atomic force microscope as a tool for structural molecular biology

Trinick/Knight - Shape changes in myosin molecules during ATPase and regulation of activity

Trinick/Kinght - Flexibility and interactions of titin

Warriner - Peptide templated synthesis of oligosaccharides

Westhead - A database of fibrilogenic mutations and experimental conditions

Westhead - Derivation and refinement of global sequence motifs for the intergal membrane proteins

Westhead - Investigation of SPNs and sequence segments of low composition complexity in genomic DNA and protein sequences

Westhead - Protein surface description and function

Westhead - Protein topology and structural alignment

Astbury Seminars 2001