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2013 CCPN/UK Leicester abstracts

Abstracts of meeting presentations

NMR with Multiple Receivers

Ēriks Kupče Bruker Ltd, Coventry, United Kingdom.

We show two main approaches for recording multi-receiver experiments - parallel and sequential acquisition. In both cases the magnetization is typically split into different pathways that are manipulated separately, with the resultant signals from each of the paths recorded using different receivers.

The sequential acquisition method has been exploited in a number of applications to date involving a range of different molecules, from small molecule to biomolecular applications. In labeled biomolecules, e.g. proteins the weak signal that remains after C-13 detected experiments (the C-13 “afterglow”) can still be measured with high sensitivity by proton detection. This is illustrated by the combined dual-receiver 2D (HA)CACO / 3D (HA)CA(CO)NNH experiment, with the latter recorded in an enhanced sensitivity mode.

While sequential acquisition has the advantage of optimizing the sensitivity of less sensitive nuclei, it suffers from modest limitations in spectral resolution. The utility of parallel acquisition is established through the introduction of the parallel 1H and 13CO detected 2D HSQC and 3D HNCA experiments in which pairs of 2D and 3D spectra are recorded in parallel.

The potential of combining parallel-receiving, multi-nuclear technologies with ultra-fast spatial encoding methods is demonstrated by the parallel 2D 1H-1H and 1H-X (X = 19F, 31P) correlation spectra acquired within a single scan. The experiment brings new opportunities for high-throughput analyses, chemical kinetics, and fast experiments on metastable hyperpolarized solutions.

With the anticipated further increases in cryogenic probe sensitivity it is expected that multiple receiver experiments will become an important approach for efficient recording of NMR data.

Sample volume in metabolomic NMR: determination and consequences

TJ Ragan, NIMR Mill Hill

The metabolomic analysis of biofluids via NMR falls into one of two categories:  either 'bottom-up' methods, where concentrations of individual components are determined and then compared; or 'top-down' methods, where overall trends in the data are detected and the spectral components contributing to those trends are then analyzed.

Bottom-up methods of analysis begin with the determination of the concentrations of various compounds in an NMR sample.  This is typically done by comparison of the signal strength with a spectral library of common metabolites.  The results from this comparison are then normalized to an internal standard and the concentrations of metabolites in the NMR sample are established.  In the case where the volume of biofluid used to prepare the sample is known, the absolute concentrations of metabolites can then be compared.  In some systems, however, a priori knowledge of biofluid volume is unavailable.  We present a method for determination of original sample volume using two standards, and apply it to the analysis of Drosophila larvae hemolymph, present in nanolitre quantities and difficult to quantify directly. 

Furthermore, we show that our method outperforms the most commonly used statistical normalization methods for top-down analyses, such as principal component analysis.

 

Deciphering complex metabolic networks using NMR spectroscopy

Christian Ludwig, University of Birmingham.

Metabolomics and metabolic flux analysis (MFA) are increasingly used to study metabolic pathways. NMR spectroscopy together with the use of selectively 13C labelled metabolic precursors is a powerful tool to study intracellular metabolic pathways. Using this approach the fate of individual carbon can be followed revealing the metabolic pathways involved in metabolisation of the 13C enriched precursors fed to the cells. However due to the complexity of metabolic networks (e.g. linear vs branched vs cyclic metabolic pathways) the resulting labeling patterns are very complicated and difficult to interpret. This problem is exacerbated by the presence of hundreds of metabolites in complex mixtures such as cell extracts.
Using high resolution 1H,13C-HSQC and TILT-TOCSY-HSQC NMR spectra together with quantum-mechanical spin-system simulations the experimental HSQC spectra can be analysed quantitatively. Once the labeling patterns are revealed, this information can be used to extract information on activity of the metabolic pathways. 
This talk will describe the experimental and computational approach used to analyse cell extract samples from the MCF7 breast cancer cell line to study glycolysis, pentosephosphate pathway and TCA cycle activity using the Matlab based software NMRLab/MetaboLab.

 

Screening protein - single stranded RNA complexes by NMR spectroscopy for structure determination

Jaelle Foot, University of Leicester

The talk will detail all the preliminary experiments performed on 2 systems to identify the suitable protein and RNA constructs and well as the buffer conditions for a structure determination of protein-RNA complexes.

 

Transient structure and dynamics of HDAC2:  How does an intrinsically disordered domain regulate protein function?

Andrea Sauerwein, UCL

Histone deacetylases (HDACs) are modification enzymes that catalyse the removal of acetyl groups from the lysine ε-amino group. Their action is
balanced by histone acetyl transferases (HATs) which add acetyl groups to the lysine ε-amino group. This interplay between HDACs and HATs  makes them
key players in eukaryotic gene expression. Whilst HDACs regulate other proteins, they are also regulated themselves by means of subcellular
localisation, association  with other proteins into multisubunit complexes, and posttranslational modifications. In human histone deacetylase 2 (HDAC2) many posttranslational modifications are located in the mainly intrinsically disordered C-terminal domain – known as the regulatory domain. To understand how this intrinsically disordered domain (150 AA) can regulate protein function a detailed knowledge of its structure in the absence and presence of posttranslational modifications is essential. Following  a challenging chemical shift assignment transient structural elements and transients interactions were quantified using a combination of chemical shifts, relaxation experiments, and spin-labelling. Of particular interest is that, when a virus phosphorylation of the C-terminal HDAC2 tail is mimicked by site-directed mutagenesis, significant changes are observed in the transient long-range interactions of this very dynamic species.
This talk will give details on the challenges of expressing an intrinsically disordered protein followed by details on chemical shift assignments and
experiments required to obtain the transient structure of an intrinsically disordered domain.

 

A multi-disciplinary approach to unravelling the Talin Rod

Ben Goult (University of Leicester)

Talin is a large, 2541 amino acid, dimeric protein that plays a key role in regulating cell adhesion and migration. Talin contains a head region that is linked to a ~220kDa flexible rod made up of 62 amphipathic helices. This rod region has a large number of binding partners including RIAM, vinculin, actin, integrin, synemin and also talin, each binding to different regions. As such, knowledge of the domain structure of the rod is essential to the understanding of talins complex role.
Using NMR, in conjunction with CD, SAXS and X-Ray Crystallography we have determined the correct domain boundaries of the talin rod. It contains 14 domains, comprising 1, 4, 5 and 9 helix bundles arranged in a largely linear fashion. This has enabled us to complete the structures of all 18 domains of talin and has allowed us to build a model of full-length talin (Goult et al. JBC 2013).
Interestingly, talin can exist in a compact, auto-inhibited state. Using our knowledge of the domain architecture in conjunction with NMR, SAXS and Electron Microscopy we have been able to produce a model of auto-inhibited, full-length talin that provides novel insights into talin regulation (Goult et al. JSB 2013).
This talk will describe the iterative approach that we employed to resolving this large and complex system with focus on the techniques and strategies employed.

 

Characterisation of the ferredoxin/IscS complex by hybrid methods

Robert Yan, NIMR Mill Hill

The bacterial ISC operon is an essential machine highly conserved from bacteria to primates and responsible for iron-sulphur cluster biogenesis. Among its components are the desulphurase IscS that provides sulphur for cluster formation, and a specialised ferredoxin whose hypothetical role is to provide electrons for the reduction of sulphur to sulphide. We have characterised the interaction between IscS and ferredoxin using a combination of biophysical tools. We used experimentally derived restraints from NMR and mutagenesis to model the ferredoxin/IscS complex using HADDOCK in parallel with SAXS and show that ferredoxin sits in a cavity close to the enzyme active site. Furthermore our data indicate competition between different components of the ISC pathway highlighting the transient nature of the protein-protein interactions involved in iron-sulphur cluster biogenesis. Our data provide the first structural insights into the role of Fdx in cluster assembly.

 

Structural characterisation of surface proteins promoting host colonisation and biofilm formation

Andrew Brentnall (University of York)

Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) are major causes of healthcare associated infections that affect millions of patients worldwide each year. Carriage of staphylococci in the anterior nares increases the risk of infection when a patient is immunocompromised. Treatment of S. aureus and S. epidermidis infections is further complicated by their tendency to form biofilms, particularly following the implantation of prosthetic devices. The homologous cell-wall anchored surface proteins SasG and Aap from S. aureus and S. epidermidis, respectively, have been implicated in adhesion to host epithelia and promoting cell accumulation in biofilms. However, the molecular basis of each process is not well understood. Using a number of biophysical techniques, including NMR spectroscopy, we have been able to identify an L-type lectin domain within SasG and Aap, potentially providing insight into how the proteins carry out their functions.