CENTRE FOR Centre for Biomolecular Spectroscopy BIOMOLECULAR SPECTROSCOPY The Centre for Biomolecular Spectroscopy aims to underpin basic medical science at King’s by providing state-of-the-art biophysical tools and expertise to the King’s research community. The Centre was established with a Capital Award from the Wellcome Trust and inaugurated on 9 September 2011 by the Principal of King’s College London, Professor Sir Richard Trainor. Major refurbishment and expansion of the NMR Facility was undertaken in 2017 The Inaugural Symposium was attended by more than 175 scientists from the UK and further afield and included presentations from Prof. Chris Dobson FRS, members of the scientific advisory board and several King’s researchers.
CENTRE FOR Centre for Biomolecular Spectroscopy BIOMOLECULAR SPECTROSCOPY How can biophysical techniques contribute to basic medical research? Understanding at the molecular level of processes in normal and disease states. Characterisation of biomolecules, their structures, interactions and functions: - kinetic and thermodynamic parameters of interactions - atomic level description of structure, dynamics and interactions Design of novel means of controlling biomolecular function for therapeutic purposes. + !
CENTRE FOR Outline BIOMOLECULAR SPECTROSCOPY Techniques and equipment Isothermal titration calorimetry - ITC Surface plasmon resonance - SPR (Biacore) Optical spectroscopy - CD + High resolution protein mass spectrometry Nuclear magnetic resonance - NMR Examples from current research Interactions of human La protein with RNA - Sasi Conte (Randall) Self-association of pH responsive peptides / gene transfer - James Mason (IPS) Investigation of slow dissociation of IgE from Fc ε RI - Jim McDonnell & Brian Sutton (Randall)
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Isothermal titration calorimetry - ITC Measurement of thermodynamic parameters of interaction: Isothermal titration calorimetry (ITC) is the only technique that can directly measure the binding energetics of biological processes, including protein-ligand binding, protein- protein binding, DNA-protein binding, protein-carbohydrate binding, protein-lipid binding and antigen-antibody binding. ITC has the ability to determine precisely the Gibbs energy ( Δ G), enthalpy ( Δ H), entropy ( Δ S), and heat capacity ( Δ C p ) changes associated with binding events. Reference: Velásquez-Campoy et al. (2004) Curr. Protoc. Cell Biol. 17.8.1-17.8.24
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Isothermal titration calorimetry - ITC Measurement of thermodynamic parameters of interaction: GE Healthcare MicroCal iTC200 The iTC200 instrument represents the latest generation of isothermal titration calorimetry (ITC) instruments. ITC has traditionally had very demanding sample requirements, but the iTC200 requires about ten-fold less material than the previous generation of ITC instruments: 300 μ L at 50 μ M : 60 μ L at 500 μ M
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Surface plasmon resonance - SPR (Biacore) Measurement of kinetic parameters of interaction - k on , k o fg , K d Understanding binding a ffj nities and interaction kinetics and thermodynamics can provide invaluable insights into the mechanisms of protein-ligand interactions.
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Surface plasmon resonance - SPR (Biacore) Measurement of kinetic parameters of interaction - k on , k o fg , K d Understanding binding a ffj nities and interaction kinetics and thermodynamics can provide invaluable insights into the mechanisms of protein-ligand interactions. dissocia&on(rate ( associa&on(rate ( Typically(a(set(of(different(ligand(( concentra&ons(are(run(
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Surface plasmon resonance - SPR (Biacore) Measurement of kinetic parameters of interaction GE Healthcare Biacore T100 The Biacore T100 instrument is a versatile biophysical tool for characterizing molecular interactions, enabling real-time and label-free binding studies. The exceptional sensitivity of the T100 system also allows interaction studies of low molecular weight compounds. Typically, about 1 ml of protein at 10 - 100 μ g/ml is required. For the analytes, typical injection volumes would be 20 - 100 μ L with concentration dependent on the expected a ffj nity.
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Optical spectroscopy - determination of secondary structure in polypeptides - molecular chemistry of proteins, peptides, DNA - absolute stereochemistry - solution state binding and interaction studies - physicochemical parameters (pH, temperature) - kinetics pH titration of a linear 25 amino acid peptide
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Optical spectroscopy - a research facility for the measurement and interpretation of chiroptical (optical activity) data - Applied Photophysics Ltd. Chirascan and Chirascan Plus instruments, conceived in collaboration with Dr. Alex F. Drake, are multimode spectrometers capable of measuring absorption, fluorescence and light scattering properties for ordinary, linearly and circularly polarised light for the same sample over the 170-1200 nm region. - applications include the assignment of absolute stereochemistry, the analysis of biopolymer conformation, quality control of biologicals, the study of molecular interactions and 1 ms stopped-flow kinetics.
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Protein mass spectrometry High resolution mass spectrometry of intact proteins Bruker MaXis mass spectrometer Mass spectrometry is an essential tool for any protein characterization facility. The Bruker MaXis mass spectrometer o fg ers ultrahigh resolution Qq-TOF technology, combining high sensitivity with exceptional mass accuracy. The instrument supports many projects in the Centre, from routine quality control of proteins undergoing structural studies, to detailed analyses of post-translational modifications and H/D exchange, as well as a number of proteomics applications. http://www.i-mass.com/guide/tutorial.html
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Nuclear magnetic resonance spectroscopy - NMR Atomic level description of structure, dynamics and interactions of biological macromolecules from a useful spectroscopic technique ... protein polypeptide nucleus atom ... to a powerful method for characterising macromolecular function
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Nuclear magnetic resonance spectroscopy - NMR - state-of-the-art nuclear magnetic resonance spectrometers equipped to address a range of problems of biological interest. - Bruker NMR spectrometers operating at 400, 600, 700 and 800 MHz enable high resolution studies of the structure, dynamics and interactions of biological macromolecules and and high throughput studies of biological fluids. - a set of specialised probes on the 400 and 600 MHz spectrometer permit studies of samples over a range of states, including lipid environments. - support for researchers by maintaining a pool of expertise and providing training and assistance as required.
CENTRE FOR Techniques & Equipment BIOMOLECULAR SPECTROSCOPY Nuclear magnetic resonance spectroscopy - NMR Atomic level description of structure, dynamics and interactions of biological macromolecules Characterisation of state of folding, oligomerisation; optimisation of domain boundaries Three-dimensional structure determination of proteins, protein domains, complexes Characterisation of protein dynamics Metal binding properties Monitoring interactions; screening of drug candidates Metabolomics Solid-state analysis + !
CENTRE FOR Some applications of NMR BIOMOLECULAR SPECTROSCOPY Folded or unfolded? hTCTP - 19.6 kDa - folded TIF-2 fragment - ~18 kDa - unfolded Simple 1 H NMR spectrum can indicate whether the protein is folded, pure, monomeric
CENTRE FOR Some applications of NMR BIOMOLECULAR SPECTROSCOPY Folded or unfolded? 110 110 115 115 120 120 125 125 130 130 10.00 10.00 9.00 9.00 8.00 8.00 7.00 7.00 1 H- 15 N HSQC spectra can reveal whether the protein is folded, monodisperse
CENTRE FOR Some applications of NMR BIOMOLECULAR SPECTROSCOPY Interactions of intrinsically disordered proteins δ 15N (ppm) B 110.0 Gly 110 110 110 110 115.0 Ser/Thr 115 115 115 115 120.0 120 120 120 120 CT 125.0 125 125 125 125 δ 1H (ppm) 9.0 8.0 7.0 6.50 6.50 9.00 9.00 8.50 8.50 8.00 8.00 7.50 7.50 7.00 7.00 6.50 6.50 Cross-peaks are lost from the spectrum of the transcription factor interaction domain in the presence of nuclear receptor ligand binding domain. Qualitative analysis is possible even without resonance assignment.
CENTRE FOR Some applications of NMR BIOMOLECULAR SPECTROSCOPY Definition of domain boundaries PDZ1 PDZ1 PDZ1 A C-terminal extension to the canonical domain boundaries is required for successful expression and purification. N- terminal extensions are required for high quality spectra of a monodisperse protein.
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