Centre for Biomolecular Spectroscopy BIOMOLECULAR SPECTROSCOPY The - - PowerPoint PPT Presentation

CENTRE FOR BIOMOLECULAR SPECTROSCOPY

Centre for 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 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 BIOMOLECULAR SPECTROSCOPY

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Outline

Isothermal titration calorimetry - ITC Surface plasmon resonance - SPR (Biacore) Optical spectroscopy - CD + High resolution protein mass spectrometry Nuclear magnetic resonance - NMR

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Techniques and equipment 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 FcεRI - Jim McDonnell & Brian Sutton (Randall)

Techniques & Equipment

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 (ΔG), enthalpy (ΔH), entropy (ΔS), and heat capacity (ΔCp) changes associated with binding events.

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Isothermal titration calorimetry - ITC

Reference: Velásquez-Campoy et al. (2004) Curr. Protoc. Cell Biol. 17.8.1-17.8.24

Techniques & Equipment

Measurement of thermodynamic parameters of interaction:

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Isothermal titration calorimetry - ITC

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 μL at 50 μM : 60 μL at 500 μM GE Healthcare MicroCal iTC200

Measurement of kinetic parameters of interaction - kon, kofg, Kd

Understanding binding affjnities and interaction kinetics and thermodynamics can provide invaluable insights into the mechanisms of protein-ligand interactions.

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Surface plasmon resonance - SPR (Biacore)

Techniques & Equipment

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Surface plasmon resonance - SPR (Biacore)

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Techniques & Equipment

Measurement of kinetic parameters of interaction - kon, kofg, Kd

Understanding binding affjnities and interaction kinetics and thermodynamics can provide invaluable insights into the mechanisms of protein-ligand interactions.

Measurement of kinetic parameters of interaction

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Surface plasmon resonance - SPR (Biacore)

Techniques & Equipment

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 μg/ml is required. For the analytes, typical injection volumes would be 20 - 100 μL with concentration dependent on the expected affjnity. GE Healthcare Biacore T100

• 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

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Optical spectroscopy

Techniques & Equipment

pH titration of a linear 25 amino acid peptide

• 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.

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Optical spectroscopy

Techniques & Equipment

High resolution mass spectrometry of intact proteins

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Protein mass spectrometry

Techniques & Equipment

Mass spectrometry is an essential tool for any protein characterization facility. The Bruker MaXis mass spectrometer ofgers 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 Bruker MaXis mass spectrometer

Atomic level description of structure, dynamics and interactions of biological macromolecules

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Nuclear magnetic resonance spectroscopy - NMR

Techniques & Equipment

from a useful spectroscopic technique ... ... to a powerful method for characterising macromolecular function nucleus atom polypeptide protein

• 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.

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Nuclear magnetic resonance spectroscopy - NMR

Techniques & Equipment

Atomic level description of structure, dynamics and interactions of biological macromolecules

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Nuclear magnetic resonance spectroscopy - NMR

Techniques & Equipment

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

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Some applications of NMR

Folded or unfolded?

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hTCTP - 19.6 kDa - folded TIF-2 fragment - ~18 kDa - unfolded

Simple 1H NMR spectrum can indicate whether the protein is folded, pure, monomeric

Some applications of NMR

Folded or unfolded?

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1H-15N HSQC spectra can reveal whether the protein is folded, monodisperse

7.00 8.00 9.00 10.00 110 115 120 125 130 7.00 8.00 9.00 10.00 110 115 120 125 130

Some applications of NMR

Interactions of intrinsically disordered proteins

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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.

6.50 110 115 120 125 6.50 110 115 120 125 6.50 7.00 7.50 8.00 8.50 9.00 110 115 120 125 6.50 7.00 7.50 8.00 8.50 9.00 110 115 120 125

9.0 8.0 7.0

B

110.0 115.0 120.0 125.0 δ15N (ppm) δ1H (ppm)

Gly Ser/Thr CT

Some applications of NMR

Definition of domain boundaries

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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.

PDZ1 PDZ1 PDZ1

Some applications of NMR

Structure, dynamics and interactions of proteins

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0.5 1 1.5

A) B) β1 β2 β3 α1 β4 α2 β5 β1 β2 β3 α1 β4 α2 β5

β1

28:-0 29:-0 3 1 :

• 2

11:7

10:7 22:91 42:32

3 9 : 3 6 4 5 : 3

55:51 56:52

3 4 : 7 7

83:79

86:82

90:22 91:22

45:30 39:36

8 5 : 8 1 8 7 : 8 3

50:47 53:50 54:50 75:72

8 2 : 7 8 23:26 3 3 :

• 4

4 : 3 4

47:28

• the N-terminal extension to canonical

domain boundaries is an integral part of the tertiary structure.

• peptide binding is associated with a

change in dynamics at the C-terminus.

• changes in hydrogen bond strength

throughout the domain may be associated with signal propagation/ allostery

J(0) (s.rad-1) J(ωN) (s.rad-1) J(ωH+ωN) (s.rad-1) x 10-9 x 10-10 x 10-11

10.0 8.0 6.0 4.0 2.0 0.0 0.0 0.0 1.0 2.0 3.0 4.0 5.0 1.0 2.0 3.0 4.0 5.0

A) B) C)

20 40 60 80 100 120

sequence

β1 β2 β3 α1 β4 α2 α5

G13 T18 F3 M-1 T30 V32 K44 S45 D49 N68 D69 G74 V81 Q85 S86 E96 L103 L105 V115 A119

Metabolomics

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• identification of biomarkers for diagnostic tests
• characterisation of physiological responses
• indicators of risk factors
• sample don’t have to be proteins: we can look at blood, urine, tissue, faeces, saliva,…
• simple 1H NMR experiments
• automation of up to 500 samples

Examples from current research

Interactions of human La protein with RNA - Sasi Conte (Randall)

La protein is a key player in the metabolism, maturation, processing, folding and sub-cellular localisation of regulatory non- coding RNA precursors. La protects 3′ ends of newly synthesised RNA pol III transcripts from exonuclease cleavage. Highly conserved N-terminal La module (La motif + RRM1) binds with high specificity to 3′ oligoU sequences.

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Martino et al. (2012) Nucl. Acids Res., 40, 1381-1394.

Examples from current research

Interactions of human La protein with RNA - Sasi Conte (Randall)

In the cytoplasm, La interacts with an array of difgerent mRNAs, including the IRES domain IV of hepatitis virus C RNA.

• HVC RNA recognition quite distinct from mode of binding to 3′ oligoU
• ITC measurements define a role for RRM2 in domain IV binding; Mg2+ decreases affjnity 7-fold

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Examples from current research

Interactions of human La protein with RNA - Sasi Conte (Randall)

• CD spectra measured as a function of temperature

support the supposed secondary structure of the RNA

• the minimal element of domain IV required for La

interaction encompasses the lower stem flanked by a single-stranded extension on either the 5′ or 3′ end

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Examples from current research

Interactions of human La protein with RNA - Sasi Conte (Randall)

In the cytoplasm, La interacts with an array of difgerent mRNAs, including the IRES domain IV of hepatitis virus C RNA.

• Chemical shift changes map the sites of interaction: mode of binding of RRM2 is non-canonical

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3′ oligoU

Examples from current research

Self-association of pH responsive peptides - gene transfer - James Mason (IPS)

Cationic, secondary amphipathic peptides for delivery of therapeutic siRNA. Design of pH-responsive peptides, involving histidine residues, to exploit pH changes that accompany endocytosis: robust and increased delivery over non-pH responsive peptides. Increased His content was expected to promote peptide release from the peptide/nucleic acid complex, improve disordering

• f anionic lipids in membranes and lead to enhancement of nucleic acid delivery. Only modest improvements were obtained.

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Investigate:

• interactions with nucleic acids
• disordering of membranes
• self-association in solution

as a function of pH correlate with results of nucleic acid delivery assay

• set of 10 peptides in which the histidine residues describe an angle of 80°
• identical nominal charge at pH 7
• sequence variation to change hydrophobicity/hydrophilicity
• introduction of proline to interrupt helical conformation

Iacobucci et al. (2012) Biochim. Biophys. Acta, 1818, 1332-1341.

Examples from current research

Self-association of pH responsive peptides - gene transfer - James Mason (IPS)

• 1. CD spectra measure secondary structure and self-association as a function of pH. As pH is lowered, dissociation is

accompanied by loss of helical conformation. Greater hydrophobicity promotes self-association and leads to lower pKa.

• 2. Proline restores an elevated pH response which correlates with pKa values of histidine residues.
• 3. Half as much peptide binds to DNA or siRNA at acidic pH as at neutral pH - greater release expected during endocytosis.

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1 2 3

Examples from current research

Self-association of pH responsive peptides - gene transfer - James Mason (IPS)

• 4. 2H echo spectra of deuterated lipids incorporated in neutral or anionic membranes reveal difgerent degrees of disorder

promoted by difgerent peptides, as a function of pH.

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Biophysical measurements provide the basis for understanding efgects of peptide sequence on nucleic acid delivery. The efgect of a more acidic pH response on peptide self-association was notable. “... strategies that seek to promote a conformational change at a less acidic pH may succeed in increasing both the effjcacy and selectivity of gene transfer”

Examples from current research

Investigation of slow dissociation of IgE from FcεRI - Jim McDonnell & Brian Sutton (Randall)

Complementing crystallographic studies, thermodynamic analysis by SPR has shown that the interaction is entropically driven.

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The presence of the Cε2 domain alters the thermodynamic parameters from enthalpically to entropically driven, through ‘preordering’ of the intrinsically unstable Cε3 domain. Holdom et al. (2011) Nat. Struct. Mol. Biol., 18, 571-576.

Contact details

Centre for Biomolecular Spectroscopy: Isothermal titration calorimetry (ITC): Surface plasmon resonance (SPR, Biacore): Protein mass spectrometry: Optical spectroscopy: Nuclear magnetic resonance (NMR): Director: Prof. Brian Sutton

• Dr. Sasi Conte
• Prof. Jim McDonnell
• Prof. Jim McDonnell
• Dr. Alex Drake, Dr. Tam Bui
• Dr. Andrew Atkinson
• ext. 6423
• ext. 6194
• ext. 6970
• ext. 6970
• ext. 8450 / 6066
• ext. 6887

Who do I contact?

CENTRE FOR BIOMOLECULAR SPECTROSCOPY

Questions

How do I apply to use the Centre’s facilities?

CENTRE FOR BIOMOLECULAR SPECTROSCOPY

There is no one route into the Centre! You can:

• contact one of the names given for a particular technique (CD, NMR, ...)
• talk to the Director
• establish a collaboration with a PI with overlapping research interests (e.g. allergy, cardiovascular)
• discuss your research issues with someone near you already using one or more of the techniques

Sasi Conte; Jim McDonnell; Mark Pfuhl; Roberto Steiner; Mark Sanderson; Matthias Gautel; Brian Sutton; James Mason; Rivka Isaacson; Jane Cox; Catherine Williamson; …

How do I pay for using the Centre’s facilities?

There are established charges for each component of the Centre. Ask for more details. BUT - please don’t be put ofg by financial considerations - first come to see whether we can help your research.