The genes for protein production are derived from natural or synthetic sources. For example the synthetic HIV proteinase gene was produced by annealing oligonucleotides produced on our DNA synthesizer and purified by reverse phase FPLC chromatography. Oligonucleotides with mismatch bases are used in site-directed mutagenesis reactions using a variety of methods including the polymerase chain reaction in the thermal cycler. We analyze cloned DNA by agarose gel electrophoresis and dideoxy chain termination sequencing. Large scale plasmid and phage preparations are run on a CsCl gradient generated by the Beckmann ultra- centrifuge. Production runs of micro-organisms expressing a recombinant protein can be grown in one of the three 10 litre fermenters available in the Department. Levels of protein production are monitored by biological or immuno assay, SDS polyacrylamide gel electrophoresis and by Western blotting. For harvesting the grown cells we use one of the 6 litre centrifuges. The early stages of downstream processing may require the passage through the Pellicon tangential flow concentrator and subsequent liquid chromatography using the fast Pharmacia Biopilot system.
These facilities have allowed us to clone several natural and synthetic genes, undertake DNA sequencing, synthesize oligonucleotides, perform site-directed mutagenesis and produce milligram amounts of protein using expression vectors in different micro-organisms. Some of these proteins have been purified to homogeneity and crystallized.
Figure 2.1 Protein engineering design cycle.
The laboratory was completely renovated two years ago with grants from the Rayne and Wolfson Foundations and provides a bright and spacious working environment, well equipped to carry out most aspects of protein preparation, characterisation and crystallisation starting from tissues or micro-organisms on both large and small scale. A large cold room is available in which chromatography may be carried out with high performance supports for gel filtration, ion exchange or hydrophobic interaction (FPLC). Purifications can be conveniently monitored with rapid electrophoresis and isoelectric focusing apparatus (Pharmacia Phast system) and products examined by electro-spray mass spectrometry (VG-platform). HPLC apparatus is available for protein and polypeptide purification. Peptides and small proteins can be made using an ABI 431 automated synthesizer. Enzyme assays may be followed with spectrophotometry (absorbance or fluorescence) and liquid scintillation counting. Amino acid sequencing and ultracentrifugation can be performed in nearby laboratories of collaborators. Protein crystallisation conditions can be studied with dynamic light scattering (DP801) and crystal growth solutions quickly prepared with automated equipment.
Figure 2.2 Positive electrospray mass spec. analysis of horse heart myoglobin on Fisons VG Biotech Platform
Methods of X-ray structure of biological macromolecules are constantly undergoing changes and development. For example, a new generation of area detectors, (imaging plates, multi- wire, and TV) is required for large molecules which produce many diffraction data. Least squares refinement requires extensive computational and computer graphics resources. At Birkbeck we have a wide range of important and interesting techniques that reflect this fast developing area of science. These include:
Figure 2.3 Crystals and X-ray diffraction patterns of eye lens proteins
(iii) CRYSTAL ANALYSIS - To overcome the phase problem, many complementary methods are used including multiple isomorphous replacement with anomalous scattering (MIRAS), multiple wavelength anomalous dispersion (MAD), molecular replacement, solvent flattening, and non-crystallographic symmetry averaging. To model and refine the structure, we use Fourier syntheses, computer graphics model building including fragment fitting to electron density, refinement with restraints and constraints, (RESTRAIN) and simulated annealing incorporating molecular dynamics, (XPLOR).
Crystal structures of peptide and proteins solved at Birkbeck
Year Protein Initial Present
solved Resolution Resolution
Polypeptides:
1975 Glucagon 3.3 2.2
1975 Insulins modified at A1 2.2
1980 Pancreatic polypeptide 1.4 0.9
1986 Oxytocin 2.8 1.0
1986 Enkephalins 0.7
1991 Nerve Growth factor 2.3
1992 Serum Amyloid Protein
1994 Endothelin 2.2 2.2
Enzymes:
1976 Ribonuclease 2.5 1.5
1977 Endothiapepsin 2.6 2.1
1985 Endothiapepsin-inhibitor 2.6 1.6
1987 Ribonuclease-inhibitor 1.5 1.5
1989 Pepsin (hexagonal) 2.3 2.3
1989 Chymosin + mutant VIIIF 2.6 2.0
1989 HIV-proteinase 2.7 2.2
1990 Mucor pepsin 2.6 2.0
1991 Human renin 2.8 2.8
1991 Mouse renin 2.0 2.0
1991 Pophobilinogen Deaminase 3.0 1.9
1993 Mouse glutathione
S-transferase 2.4 2.4
1994 Fe Superoxide dismutase 2.5 2.0
1994 Elastase II 2.0 2.0
Proteins of the eye lens:
1978 B-crystallin 2.8 1.5
1985 E-crystallin 2.3 2.3
1988 D-crystallin 2.3 2.3
1989 E-crystallin rat 2.3 2.3
1990 B2-crystallin I222 form 2.6 2.1
1991 B2-crystallin C222 form 3.3 3.3
1994 B- domain 1.5 1.5
1994 -crystallin P212121 2.5 2.5
1994 -crystallin C2 4.0
1994 B-crystallin-water 150K 1.2
1994 B C-terminal domain 1.5 1.5
1994 B C-terminal domain
minus C-terminal tyrosine 3.0
Iron transport proteins:
1988 Transferrin (rabbit serum) 3.3 3.3
1989 Transferrin (N-lobe) 2.3 2.3
1989 Ovotransferrin
(N-fragment duck) 2.3 2.3
Other:
1991 Human placental annexin V 2.8 2.8
1993 Bacteriophage T4
-glucosyltransferase 2.2 2.8
The Department EM unit contains scanning and transmission electron microscopes and preparation equipment, for use by department members and also by external users of the Intercollegiate Service. The main instruments for work in structural molecular biology are a JEOL 2010 HC Cryo TEM and a JEOL 1200EX Cryo TEM. These provide the facilities for cryo EM imaging of frozen-hydrated samples, suitable for analysis of protein subunit shape and packing symmetry in membranes, viruses and oligomeric structures.
For symmetrical or ordered structures, structural information can be greatly enhanced by locating and averaging together many images of a repeated structural feature, for example the unit cell of a two-dimensional crystal of a membrane protein, or the structural units of an icosahedral virus. Three-dimensional structures are reconstructed by combining views in different orientations by computed tomography. Our image processing is done on Silicon Graphics and DEC alpha workstations using SPIDER and MRC image programs.
Current projects include work on two-dimensional crystals of the visual pigment rhodopsin, molecular chaperones and retroviral core-like particles.
3D reconstruction from cryo EM.
Neutron scattering is used in the study of both structure and dynamics of both crystalline and liquid samples at the atomic or molecular level of resolution. With the advent of research reactors, culminating in the opening of the high flux reactor at the Institut Laue Langevin (I.L.L.) in the 1970s, the specific advantages of neutron scattering could be exploited. The wide range of wavelengths available (typically 0.1 - 30 ) is appropriate for examining both atomic scale structures and large spatial correlations; in addition, the energies of the neutrons used in scattering experiments are similar to the excitation energies of the samples we wish to study. This means we can use neutrons not only to examine structure (elastic scatterings) but also the change in the energy of the neutron as it is scattered tells us about the motions inside the sample (inelastic scattering). The amplitude of scattering of neutrons and of X-rays is very different for most atoms - for instance hydrogen atoms can be 'seen' by neutrons but not by X-rays - which makes neutron diffraction a very useful complementary technique to X-ray diffraction in structure determination.
A considerable amount of work has been done in the Department using the high flux reactor source at I.L.L.. A neutron diffraction study of the vitamin B12 crystal (Savage 1986) led to a proposed new model of water interactions (Savage and Finney 1987). A very low temperature (15K) and high resolution crystallographic study has now shown the water networks within the molecule in great detail (Bouquiere, Lindley and Finney 1993, 1994). The ineteraction between water and small molecules in aqueous solution has been studied using the isotope substitution difference method (Turner et al., 1986, 1990). The I.L.L. source restarted in late 1994 after a 2-year shutdown for upgrading.
The last decade has seen rapid developments in techniques of time-of-flight neutron scattering at the ISIS neutron scattering facility at the Rutherford Appleton Laboratory in Oxfordshire. This is a source of pulsed neutrons providing high intensity and a very wide range of energies which currently has instruments designed for high intensity and/or high resolution powder diffraction, small angle scattering, liquids and amorphous materials structure and dynamics in crystalline and non-crystalline systems. Current and recent work in the Department includes studies of water structure in solutions of apolar solutes (Turner and Soper), high resolution studies of high pressure phase of ice (Londono and Finney), low energy motions of lysozyme and of the eye-lens protein gramma-crystallin (Bouquiere and Finney; Moss et al.) and the conformation and association behaviour of gamma-crystallin in solution studies by low-angle scattering (Moss, Purbhoo, Turner and Slingsby).
Figure 2.5 Neutron diffraction study of water networks in vitamin B12 at 15K.
The Department has a local area network which currently provides access to a number of workstations and supports around 40 interactive users. The main cluster consists of six VAX machines including four Evans and Sutherland (PS300, PS390, ESV10) and over 10 Silicon Graphics machines. Data collection devices (such as the FAST and two Imaging Plate area detectors) are networked.
The Silicon Graphics machines have been used for a variety of modelling applications with the QUANTA, INSIGHT, O and SYBYL software packages. The Evans and Sutherland machines are used primarily for the interpretation and modelling of electron density using FRODO and O.
Figure 2.4 Electron density fitting using graphics
We have direct access through JANET and now SUPERJANET to the CONVEX and CRAY supercomputers at ULCC, RAL and Edinburgh. These are used for intensive calculations such as molecular simulations. We also have access to other computer centres such as those at Daresbury (X-ray synchrotron) and at ISIS (neutron source) for specific projects. The Department also uses the Birkbeck College VAX 6310 and RS6000 computers.
Through the SERC Computational Science Initiative we purchased a Convex C220 minisupercomputer which is likely to be superceded during the year by a cluster of UNIX workstations. We have 4 Hewlett Packard 9000 series workstations mainly used for simulation work and a DEC Alpha to support UNIX mail and the UNIX interactive service. Although we continue to maintain our VAX cluster, we have an ever increasing number of UNIX-based workstations.
The Department has a state-of-the-art Aviv 62DS circular dichroism instrument that is fitted with a thermally controlled sample chamber, fast scan kinetics, variable position detector (for use with scattering samples such as viruses and membranes), and interfaced to the department computing network. In addition we have a Cary UV/Vis scanning spectrophotometer, which has a software for kinetics and first and second derivative spectra.
My group has written software for data reduction, processing, and analysis, including calculations of secondary structures, statistical analyses, corrections for differential absorption flattening effects, and solvent-dependent wavelength shift phenomenon.
Circular dichroism spectroscopy is being used for the determination of secondary structures, the monitoring of unfolding and refolding of proteins by temperature, pH, and chaotropic agents, and the examination of ligand- and environment-induced conformational changes in membrane proteins, soluble proteins and peptides, and viruses. Methods development work includes theoretical studies of dipole dependence of wavelength shifts, the analysis of differential scattering in large particles, and more accurate algorithms for secondary structure analysis.
Figure 2.8 Circular Dichroism spectra of the ion channel-forming polypeptide Alamethicin
being
