Protein Structure Determination using NMR Spectroscopy. Cesar Trinidad

Transcription

1 Protein Structure Determination using NMR Spectroscopy Cesar Trinidad

2 Introduction Protein NMR Involves the analysis and calculation of data collected from multiple NMR techniques Utilizes Nuclear Magnetic Resonance theory Measures resonance frequencies emitted by nuclei with spin properties

3 Theory of NMR Atomic nuclei spin Found in atoms with odd number of protons/neutrons Nuclei acts like a small magnet that resonates at a particular frequency relative to its environment Most commonly used NMR active nuclei 1 H, 13 C, 15 N

4 Instrumentation Sample holder RF Transmitter Receiver Magnet Data Recorder

5 Helium gas is used to cool the superconductive magnets (below 10 K) Liquid nitrogen is used to cool down the Helium (reduces He loss)

6 Principles

7 Sample Preparation Pure protein samples Overexpression using E. coli Purification i.e. affinity tags, immunoprecipitation Increases resolution Stable isotope labelled proteins Bacterial expression i.e. E. coli in C 13 carbon source Provides ample quantities of uniformly isotope protein required by NMR spectroscopy Protein must not degrade during experiment Multidimensional assays require days or weeks of measurements

8 Isotope labelling Heteronuclear NMR Requires coupling of different isotopes Most Common isotopes used in Heteronuclear NMR 2 H, 13 C, 15 N

9 Sample Preparation: Solvents Deuterated solvents Removes spectral overlap caused by the solvent Deuterochloroform (CDCl 3 ) non-polar D 2 O and deuterated DMSO (DMSO-d 6 ) polar Compound Mass (g/mol) Deuterated Mass CHCl CDCl H 2 O D 2 O DMSO DMSOo-d Deuteration of compounds are rarely 100%, therefore, residual resonance from 1 H is observed

10 Principles

11 Resonance Assignments Involves multiple NMR experiments to determine resonance between molecules 1. Backbone assignments Resonance measurements of isotopes in backbone relative to each other 2D 15N-HSQC: First set of HN resonances used as reference for future measurements 2. Residue assignments

12 2D 15N-HSQC Also used to check for protein folding

13 Backbone Assignments Other NMR experiments: - HBHAcbcacoNH - HNcaHA

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15 Side chain Assignments Once sequential connection is identified, data from HA, HB, CA and CB resonances are used for assigning side chains to backbone

16 Standard H and C NMR chemical shifts in protein

17 Principles

18 Data from multiple backbone, sidechain and NOE assignments are analyzed to determine secondary structure

19 Principles

20 NOESY Nuclear Overhauser effect Used to determine distances between molecules even without covalent bonding In 3D NOESY, calculations are done by automated assigning softwares: AUDANA, PONDEROSA accepts input information consisting of a protein sequence, backbone and sidechain NMR resonance assignments, and 3D- NOESY spectra

21 Structure calculations

22 Applications

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24 Determining binding affinity 2D-HSQC experiment can be used to characterize the strength of ligand binding Position and intensity of cross peaks changes overtime Peak of free protein reduces and new peak from the complex is formed

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26 Features of Protein NMR Observe conformations of proteins Observe protein dynamics and mobility Study protein folding, conformational changes Identify ligand binding sites and affinities Assay conditions can be performed under biological conditions (ph, ionic strength etc)

27 Limitations Structural determinations are very slow (may take days weeks) Instruments are expensive Helium for cooling

28 Future Improvements Increasing the resolution of the spectras collected Stronger magnets Better detector probes Spectral overlap limits the size of protein analyzed Size limit for NMR spectroscopy used to be less than 50kDa but recent advances allows 150kDa

29 Conclusion Protein NMR allows complete visualization of protein stucture Allows in depth characterization of protein properties based on structure e.g. binding sites, folding patterns, conformations. Applications Medicine (drug design) Protein design Proteomics (protein database)

30 Bibliography Determination of protein structures in solution using multidimensional heteronuclear NMR spectroscopy. (1993). Toxicon, 31(5), Downing, A. (2010). Protein NMR techniques. Totowa, N.J.: Humana Press. Li, Y., & Kang, C. (2017). Solution NMR Spectroscopy in Target-Based Drug Discovery. Molecules, 22(9), Skinner, A., & Laurence, J. (2008). High-field solution NMR spectroscopy as a tool for assessing protein interactions with small molecule ligands. Journal Of Pharmaceutical Sciences, 97(11),

31 Questions