Dr Ashley M. Buckle

Senior Research Fellow

Telephone: +61 3 990 29313 Facsimile: +61 3 990 29500 Office: Room 206, Building 77, Clayton Email: Ashley.Buckle@med.monash.edu.au

Research interests

My research is split between structural studies of a variety of interesting biological systems (using protein crystallography), and bioinformatics. A selection of current projects is given below.

Find out more about proteins at the PDB and NIH .

Research Group Members

Dr Kate F Fulton (NHMRC Peter Doherty PostDoctoral Fellow)
Dr Chris Betts (Senior Research Fellow)
Mr Mark Bate
Mr Abdullah Amin

Structural Biology

Serpins
In collaboration with Dr's James Whisstock, Steve Bottomley and Rob Pike (Monash University).

Serpins (serine protease inhibitors) fold into a native metastable state and utilize a complex conformational change to inhibit target proteases.   An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperature. We have recently determined the 1.8 Å X-ray crystal structure of thermopin, a thermophilic serpin from Thermobifida fusca.  We reveal the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures (Buckle et al, 2005). [PDB entry 1SNG].We have determined the X-ray crystal structure of Maspin, a serpin that acts as a tumour suppressor in a range of human cancers, including tumours of breast, prostate and lung.  Structural analysis has allowed us to highlight putative cofactor-binding sites on Maspin, as well as a region of striking conformational flexibility.  This work provides a detailed molecular framework to elucidate the mechanism of function of this important human tumour suppressor (Buckle et al, 2005). [PDB entries 1WZ9, 1XU8]. Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. However, the Myeloid and Erythroid Nuclear Termination stage-specific protein (MENT) is also a non-histone architectural protein, and participates in DNA and chromatin condensation. In an effort to understand better how MENT marries serpin activity with chromatin condensation, we have recently determined the X-ray crystal structures of wildtype and variant MENT in native and cleaved conformations, and investigated how the conformational change and polymerization of MENT plays a fundamental role in the physiological process of chromatin condensation (McGowan et al., 2006) [PDB entries 2H4P; 2H4Q, 2H4R; 2H4S].

Proteases
We have recently embarked on structural studies of inactivated protease paralogues from the scabies mite Sarcoptes scabiei, in collaboration with Katja Fischer and David Kemp (QIMR) Find out more about the structural biology of proteases. See a flash animation of the protease mechanism.

Transthyretin-Like Proteins (TLPs)
Transthyretin is a tetrameric protein that is responsible for transport of thyroxine and related molecules, and is structurally well characterised. Transthyretin-like proteins (TLPs) are a family of homologous proteins present in a large range of bacterial, fungal, plant, invertebrate and vertebrate species. TLP's form tetramers yet do not bind thyroid hormones. In order to understand their biological role we have determined the crystal structure of the TLP from the gram-negative bacterium Salmonella dublin, the first structure of a TLP to be determined (Hennebry et al.,, 2006). [PDB entry 2GPZ].

Bioinformatics: Protein Databases and Tools

In collaboration with Drs Steve Bottomley, Kate Fulton, and James Whisstock (Monash University)

REFOLD: A Database for Protein Renaturation

The majority of human proteins expressed in bacteria are insoluble and thus require renaturation.  Identifying the optimal refolding conditions and methodology is therefore rate limiting. In order to address this problem, we have catalogued the methods employed in the refolding hundreds of proteins in a web-accessible database, REFOLD. The database contains annotated entries for the refolding of a wide range of proteins, as well as detailed protocols and experimental information that may assist in the design of new renaturation protocols. (Buckle et al, 2005; Chow et al, 2006)

The Protein Folding Database (PFD) and the Foldeomics Consortium

Understanding the rules that govern protein folding is one of the great challenges of molecular biology. The last 10 years have witnessed a revolution in our understanding of the pathway and stability of protein folding, and the number of proteins being studied is growingly rapidly. In order to address this growth of data, we have created the Protein Folding Database (PFD), which collects annotated structural, methodological, kinetic and thermodynamic folding data into a single public resource. The web interface allows searching, browsing and information retrieval, and provides links to other protein databases (Fulton et al, 2004). For more information see the Wikipedia article on protein folding and Folding@Home.

CLIMS2: Crystallography Laboratory Information Management System 2

CLIMS2 is a Laboratory Information Management System for protein Crystallography that features a novel graphical interface to a relational database, and assists all aspects of protein crystallisation, including: protein expression and handling; crystallisation optimisation; visualisation of results and preliminary diffraction data (Fulton et al, 2004; Amin et al, 2005). CLIMS is in use in many laboratories worldwide.

Summary of Past Research (1990-2003)

1990-1994 PhD Chemistry, Cambridge University. Lab of Prof Sir Alan Fersht, MRC Centre for Protein Engineering, Cambridge.
1994-1996 Medical Research Council (UK) Postdoctoral Fellow, Cambridge.
1996-1999 Research Associate, MRC Centre for Protein Engineering, Cambridge.
1999-2003 MRC staff scientist, Cambridge

• Protein stability: high-resolution crystal structures of a large number of mutants of barnase, allowing us to interpret structural responses to mutation in a thermodynamic context. (Buckle et al, 1993; Buckle et al, 1996; Vaughan et al, 2002).
• Protein-protein recognition: elucidation of the crystal structure of the barnase-barstar complex revealed the structural basis for the very tight and rapid binding that characterizes the complex (Buckle & Fersht, 1994; Vaughan et al, 1999). [PDB entry 1BRS].
• Ribonuclease mechanism: the structure of the complex between barnase and a DNA substrate analog provided the most comprehensive model of ribonuclease action to date, and a detailed insight into the enzyme-transition state complex (Buckle & Fersht, 1994). [PDB entry 1BRN].
• Molecular chaperones: elucidation of the crystal structure of the minichaperone gave the first detailed structural model of GroEL-substrate interactions and a structural basis for the allosteric mechanism of intact GroEL.GroES.ATP complex (Buckle et al, 1997; Zahn et al, 1996; Altamirano et al, 1997; Wang et al, 2000, 2000; Chatellier et al, 1999). [PDB entries 1JON, 1KID]. Find out more about chaperones

Publications

[see also PubMed and Google Scholar]

A full list of Dr Ashley M. Buckle's publications Dating from 1993 - 2006