Protein NMR. Bin Huang

Transcription

1 Protein NMR Bin Huang

2 Introduction NMR and X-ray crystallography are the only two techniques for obtain three-dimentional structure information of protein in atomic level. NMR is the only technique for 3-D determination of protein in solution state. Multiple-NMR can identify the 3-D structure of proteins up to 40,000KDa. NMR is used to study the kinetic reactions of proteins.

3 Contents Protein NMR sample preparation Methodologies of protein NMR Disadvantages of protein NMR

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5 NMR sample preparation Since 2-D and 3-D NMR always takes days to finish, appropriate preparation is very important, and additives are highly recommended to add. Protein concentration in the NMR sample should be about 0.5-2mM. Samples need to be high purity, stable, and high soluble in D -solvent. anti-microbial agents: sodium azide (NaN3) at a concentration of 0.02% is recommended. EDTA or AEBSF are frequently added to reduce proteolysis at a concentration of 0.1-5mM. A buffer: phosphate buffer is always used to provide a longterm stable condition for proteins.

6 Common Methods for protein NMR Isotope Labelling : basic method for protein NMR 1-dimentional NMR : for small proteins 2-dimentional NMR: COSY, TOCSY, 2-D HSQC. 3-Dimentional NMR: HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, 3-D HSQC.

7 1-D NMR Is the routine NMR techniques for protein in the size range between 5-25KDa but have many failed examples due to protein aggregation and dynamics. The amino acid residues in proteins have specific chemical shift therefore can be used as reference

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9 Isotope Labelling Applied for almost NMR studies currently The basic purpose is to reduce the signal overlap and linebroadening Reduce NMR peak overlap: 1 H- 15 N and 1 H- 13 C HSQC spectra to resolve the 1H resonance overlap Increases signal-to-noise ratio: 2 H is less active and has 6.5times longer relaxition time of 1 H. 2 H replacement of 1 H leads to reduced dipole interactions. 13 C and 15 N enrichment also leads to strong signals. Resonance assignment: 13 C and 15 N enrichment makes the possibility to assign the sequence of the backbone and side-chain by using one bond or two-bond scalar coupling. Structural information: 2 H replacement of 1 H reduces the nuclear spin diffusion. 13 C and 15 N enrichment used to obtain the secondary chemical shifts based on NOEs.

10 Methods for Isotope Labelling Gene expression in living organisms such as E.Coli, yeast cells or other living cells. Because E.Coli. Grow rapidly and its properties are well studied and known, E.Coli is the most common used host for gene expression system. Cell-free protein production uses the E.Coli extraction which contains the protein synthesis machinary but with the metabolic system is suppressed.

11 Two types of Isotope Labelling Uniform labelling: all of the atoms are labelled without any selectivity. Uniform labelling is a low cost labelling. Selective labelling: just selected positions are isotopically labelled.

12 Uniform Labelling uniform 15 N labelling is always the initial step for protein characterization by its low cost. 15 N labelled proteins are always produced by fed E.Coli with 15 NH 4 Cl as the sole nitrogen source. uniform 15 N and 13 C labelling are always produced by fed E.Coli with 15 N labelled salts and 13 C labelled glucose. Uniform 2 H labelling are commonly produced by fed E.Coli with duterited water, 2 H 2 O.

13 Selective Labelling Amino acid Type-Selective labelling: a cell-free production system is suitable. All 20 isotopic labelled amino acids are commercially available. Segmental labelling: labels half of the protein, either N- terminus or C-terminus. SALL/stereo-array isotope labelling: labels one of the prochiral protons of methylene groups.

14 by linking the diagonal-peaks and the corresponding cross-peaks.

15 This is always the figerprinter of protein sequencing.

16 3D NMR Triple resonance hetero-nuclear correlation spectra. Coupling between 1 H- 13 C- 15 N in one amino acid residue. There are three chemical shift axes, 1 H-axis, 13 C-axis, and 15 N-axis. 1 H- 13 C- 15 N HNCA, HNCO, HN(CA)CO, HN(CO)CA, HVACO, HCA(CO)N : For sequence assignment of backbone. 1 H- 13 C- 15 N CBCA(CO)NH, HNCACB, CC(CO)NH: for side-chain assignment

17 Back-bone assignment Side-chain assignment

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21 Disadvantages Molecular weight limitation: as the mass of proteins increases, peak overlap increase but with the reduce of relaxation time. 2D Homonuclear Correlation Spectroscopy: limitation of less than 80 amino acids. 2D and 3D Homonuclear Correlation Spectroscopy: limitation of less than 200 amino acids.

22 Reference 1. Lu Yun Lian, Gordon Roberts. Protein NMR Spectroscopy: Principal Techniques and Applications. 2. Introduction to Protein NMR Structural and functional study of protein by NMR spectroscopy Determining 3D structure of proteins Nuclear Magnetic Resonance (NMR) Spectroscopy. nmr-1.html