List of Projects for TSMB '09-'10

Note: Make sure you read the paragraph describing each project carefully before making your choice. Students who produce their own clear, relevant diagrams, using appropriate software, will be marked significantly higher than those who copy equally relevant diagrams from elsewhere with due acknowledgement.

Structural Investigations of Bacterial Secretion Systems

Bacteria have to secrete proteins, particularly those pathogenic bacteria that are taken up into a host cell to get nutrients and need to subvert the cell to their own purposes. There are several different secretion systems see http://en.wikipedia.org/wiki/Secretion . The aim of this project is to assess progress on understanding the structural basis of how these systems work and to describe and comment on which structural methods have been useful in understanding them.

Structural investigations of the ribosome

One of the most important advances in structural biology has been the elucidation of the ribosome at atomic resolution and the insights this has brought to protein synthesis. Both X-ray crystallography and electron microscopy have made important contributions to these advances. Describe how each method has contributed to our current understanding of the structure and discuss some of the most recent advances which have been increasing our knowledge of accessory proteins binding to the ribosome and the full protein synthesis pathway.

Protein Solubility

Most of the protein structures in the PDB have been determined from aqueous solutions. Discuss the factors that determine protein solubility. If a recombinant protein expressed in E. coli goes into an inclusion body, what strategies can be used to gain a 3D structure? Give examples of structures that have been determined from proteins purified from inclusion bodies and the methods used as well as examples of methods used to alter the expression of proteins so that they are soluble.

Comparison of fusion protein systems for protein purification.

There are several different fusion protein systems used for protein purification. This project will involve searching the literature and the web (company web sites and catalogues will be particular useful here) to list the range available. A discussion of the advantages and disadvantages of each type of fusion protein is also required.

Fragment based Drug Design

The use of small chemical fragments that bind with low (mM) affinity to drug targets as starting models for drug design is a relatively new development. Biophysical (NMR, SPR, Calorimetry, X-ray Crystallography) are used to screen libraries of small molecules. From a weakly binding compound a series of larger compounds are designed to find a molecule with tighter binding (nM) suitable for use as a drug. Although the affinity of these small molecules is low, the ligand efficiency (affinity/molecular weight) is often very good. Amongst the companies that developed this method are Astex pharmaceuticals (X-ray) and Abbot Laboratories (SAR-by-NMR). The project will describe these approaches and some of the successes.

Attenuated total reflection (ATR) infra-red spectroscopy of proteins

The high sensitivity of modern ATR Fourier transform infra-red spectrometers and the recent development of time-dependent and equilibrium methods to obtain accurate difference spectra (notably by the groups of Klaus Gerwert and Peter Rich) are able to reveal details of changes in structure, hydration and protonation states in protein complexes as a result of changes in pH, redox potential or ligand binding. This project will outline the principles underlying these methods and survey their application and potential to advance our mechanistic understanding of protein function.

Electron Microscopy, Crystallography and Molecular Modelling

Detailed molecular structures of many large protein complexes or "molecular machines" have been produced using a combination of techniques. This is done by fitting atomic resolution structures of individual protein chains (or even domains) into the lower-resolution electron density maps from electron microscopy (or from low resolution X-ray crystallography). Describe the methods for fitting structures determined by X-ray crystallography or NMR into low resolution maps and give examples of how this has been used in practice, what biological insight it has given, and the limitations that arise from the low resolution.

Artificial construction of de novo kilobase DNA

Oligonucleotide chemical synthesis enabled the widespread adoption of PCR and various mutagenesis protocols. Since that time, techniques have appeared that make possible the de novo construction of artificial DNA sequences, which can span hundreds or even thousands of nucleotides in sequential length. Review the various methods that have appeared in the literature, and compare and contrast the value in performing the most efficient techniques in the laboratory, against the various and best known commercial options. The ultimate example of this was the artificial synthesis of a whole genome by Craig Vetner. You should also discuss the more mundane uses in structural biology of these techniques.

Methods for Automated Crystallographic Model Building

Increasingly automated methods can be used for building a nearly complete atomic model (PDB) file from an initial experimentally phased electron density map or molecular replacement solution. These programs include ARP/wARP, Bucanner (CCP4), Resolve and Textal (Phenix). The project will outline how the various automated building programs work and describe what applications the methods are effective for and their limitiations.

Measuring progress in protein structure prediction: the CASP series of structure prediction competitions.

CASP (Critical Assessment of Structure Prediction) http://predictioncenter.org/ is a series of biannual "competitions" to assess the quality of protein structure prediction methods. The results of each competition are published in a supplement to the journal Proteins and there are reviews published after each CASP conference. This project will involve reviewing the progress made in structure prediction during the first eight competitions (from 1994 to 2008), and critically discussing the most successful approaches and methodologies.

Phosphoinositide binding domains in the PDB

Phosphoinositides are one of the most important classes of cell signalling molecules. There are several types of protein domain that bind them, some specific for certain inositides while others bind inositides nonspecifically. The project should use the Protein Data Bank and the literature to describe the structures of these domains and how discrimination between different inositides is achieved.

Prevention and use of radiation damage in protein crystals

Radiation damage is a major problem with protein crystals particularly at third generation synchrotrons. The project will present an overview of the causes of radiation damage, its prevention and how it can be used to the experimenter's advantage. Work particularly by Elspeth Garman has tried to systematically understand the problem and find methods of prevention. Raimond Ravielli and colleagues have also deliberately used radiation damage to derive phase information and to study which part of the protein is damaged first.

Restriction/methylation systems and their structure and use in molecular biology

This project requires analysis of the scientific literature and on-line resources to discuss the various types of restriction/methylation systems. The aim is to give an overview of their biological role, their use in DNA manipulation methods and to discuss the relationship of structure to function for at least one example of a methylase and one endonuclease within the overall context of what is known about these enzymes from a structural perspective.

Structural Studies of Filaments

Filaments can be studied by electron microscopy and X-ray fibre diffraction. Describe these methods and discuss the biological insights that have come from them. The most notable examples are from the cytoskeleton. There are examples of both methods in section 12 which could form a starting point for this project. You should look to give examples of at least three applications of each method to different proteins.

Analysis of Protein Complexes by Mass Spectrometry

Mass spectrometry takes place in the gas phase. This and the sample preparation techniques usually mean that protein complexes are broken into their constituent peptides. However, recent modifications have allowed the masses of intact complexes to be measured. A different innovation has been to isolate large complexes from cells and use mass spectrometry to determine which proteins are in the complex. Describe how mass spectrometry has been adapted to achieve these two different types of results and compare the different types of biological insight the two methods have given.

SAXS/SANS (Small angle X-ray and neutron scattering)

Small angle X-ray and neutron scattering can be used to probe the low resolution shape, conformation and oligomeric state of macromolecules. In some cases to a similar resolution as electron microscopy. The project will outline the theory of the methods and concentrate on on at least four detailed examples where the results have given biological insight.

Tomography by Electron Microscopy TWO TAKERS ALREADY

Sectioning of vitrified cell and tissue samples has been pioneered by J Dubochet and tomography of vitreous samples by W Baumeister. Tomography of vitrified sections provides a view of cells and tissues in their native, hydrated state, without the severe artefacts of fixation, embedding in plastic resin and thin sectioning. Write a review of these important methodological developments and their significance for cell and molecular biology.

FRET (Fluorescence Energy Resonance Transfer) and FLAP (Fluorescence Localisation after Photo Bleaching) in vivo

Fluorescence techniques such as FRET and FLAP are increasingly being used to study protein interaction and mobility in intact cells. Describe these methods, giving examples of the type of studies that can be carried out with the techniques and their limitations.

Please refer to the guidelines before choosing your project.

Nicholas Keep and Clare Sansom, June 2010