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NMR Spectroscopy in Structural Biology Atia-tul-Wahab, M. Iqbal Choudhary and Kurt Wüthrich, 1 Dr. Panjwani Center for Molecular Medicine and Drug Research,

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Presentation on theme: "NMR Spectroscopy in Structural Biology Atia-tul-Wahab, M. Iqbal Choudhary and Kurt Wüthrich, 1 Dr. Panjwani Center for Molecular Medicine and Drug Research,"— Presentation transcript:

1 NMR Spectroscopy in Structural Biology Atia-tul-Wahab, M. Iqbal Choudhary and Kurt Wüthrich, 1 Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan The Scripps Research Institute, La Jolla, CA, USA

2 X-Ray VS NMR Structures Molecules are studied in solution, closer to the native condition found in cell. Protein folding studies can be done by monitoring NMR spectra. Chemical or conformational exchange, internal mobility and dynamics at timescales ranging from picoseconds to seconds. NMR is very efficient in mapping interactions with other molecules, e.g. protein/protein, protein/nucleic acid, protein/ligand or nucleic acid/ligand interactions. The upper weight limit for NMR structure determination is ~30 kDa. NMR StructuresX-Ray Structures Crystallization required, potential crystal packing influence the structure, especially on the surface of protein. Flexible loops may not be visible in crystal structure due to spatial arrangement of electron density. Above ~ 30 kDa X-Ray is the only technique to solve the structure of proteins. 2

3 The Steps in Protein Structure Determination by NMR 1.Sample preparation (a) protein selection (b) gene engineering (c) protein expression (d) protein purification (e) buffer optimization (f ) isotope labeling 2.Data collection (a) HSQC (b) amide H/D exchange (c) APSY/ triple-resonance (d) 3D-NOESY 3.Data evaluation 4.Structure calculation 5. Structure refinement 6. Structure deposition 3

4 Sebastian Hiller et al., J. Biomol. NMR (2008) Automated Projection Spectroscopy (APSY) 4

5 Fig. 2 (2003) Progress in NMR Spectroscopy, 43, 105, Guntert. The Assign Calculate Evaluate cycle Automated NOE assignment and structure calculation 5

6 Linking spin systems using nOes 6

7 1D 1 H-NMR screening Protocol for Automated NMR Structure Determination 1. NMR Sample 2. NMR Structure Promising protein constructs and solvent conditions 2D [ 15 N, 1 H]-HSQC screening Structure quality protein solution NMR profile Automated backbone assignments Interactive validation of backbone assignments Chemical shifts adaptation to NOESY spectra Interactive validation of backbone assignments Chemical shifts adaptation to NOESY spectra Automated [ 1 H, 1 H]-NOESY-based side chain assignments, constraints collection and structure calculation NMR structure solved Accurate backbone fold NMR structure solved Accurate backbone fold Interactive NMR structure refinement NMR structure refined NMR structure validation PDB No 7

8 PJ03720C TM0320 A. Folded globular protein B. Non-globular protein Folding PG9814A C.Aggregated or oligomerized protein 8

9 [ 1 H, 15 N]-HSQC Spectra TM0320 GS13720A PE00019A 9

10 Number of expected peaks Peak number (order with decreasing intensity) Signal/Noise Signal-to-Noise NMR Profile 10

11 Signal/Noise Peak number (order with decreasing intensity) APSY quality TM0320 NMR Profile Number of expected peaks 11

12 Peak Number (order with decreasing intensity) Signal/Noise 88 Number of expected peaks PE00019A NMR profile Limited APSY quality 12

13 NMR Experiments 3 APSY-NMR experiments projections each 3 APSY-NMR experiments projections each 3D- 15 N-resolved [ 1 H, 1 H]-NOESY 3D- 13 C-resolved [ 1 H, 1 H]-NOESY (ali) 3D- 13 C-resolved [ 1 H, 1 H]-NOESY (aro) 3D- 15 N-resolved [ 1 H, 1 H]-NOESY 3D- 13 C-resolved [ 1 H, 1 H]-NOESY (ali) 3D- 13 C-resolved [ 1 H, 1 H]-NOESY (aro) Automated backbone assignment Interactive validation of backbone assignment Chemical shifts adaptation to NOESY spectra Interactive validation of backbone assignment Chemical shifts adaptation to NOESY spectra Automated [ 1 H, 1 H]-NOESY-based sidechain assignment, constraint collection and structure calculation NMR structure solved Accurate backbone fold NMR structure solved Accurate backbone fold Interactive NMR structure refinement NMR structure refined 13

14 Software GAPRO MATCH ASCAN ATNOS CANDID MATCH ASCAN ATNOS CANDID UNIO Automated backbone assignments Interactive validation of backbone assignments Chemical shifts adaptation to NOESY spectra Interactive validation of backbone assignments Chemical shifts adaptation to NOESY spectra Automated [ 1 H, 1 H]-NOESY-based side chain assignments, constraints collection and structure calculation NMR structure solved Accurate backbone fold NMR structure solved Accurate backbone fold Interactive NMR structure refinement NMR structure refined CYANA 14

15 15 Protein NP_ , A Phage-Related Protein isolated from Bordetella bronchiseptica No structure was available of the whole family Function of the protein was not known C-terminal N-terminal C-terminal N-terminal

16 Number of amino acid: 141 Mol. Wt: 15.2 kDa Experiments: 4D-HACANH 5D-HACACONH 5D-CBCACONH 13 C-resolved NOESYs (ali & aro) 15 N-resolved NOESY 75.6% backbone assignments 68.4% Side chain assignments GMSQDLIRAAFEKRLSDWAKARTPALPVAWQNTKFTPPAAGVYLRAYV MPAATISRDAAGDHRQYRGVFQVNVVMPIGDGSRSAEQVAAELDALFP VNLVMQSGGLAVRVRTPISNGQPTTGDADHTVPISLGYDVQFYPE 16

17 17

18 18

19 C-terminal N-terminal Sequence CA-CB (ppm) Secondary Structure Elements 19

20 C-terminal N-terminal

21 Calculated RCS/ppm Observed CS/ ppm Experimental Chemical Shift Difference from the average of Methyl groups in Val, Leu, Ile vs Calculated Ring Current Shifs 21

22 Heteronuclear NOE Results Sequence A51 A124 G78 Q31 N-terminal C-terminal II I III IV Relative intensity 22

23 C-terminal N-terminal C-terminal N-terminal C-terminal N-terminal C-terminal N-terminal 23

24 C-terminal N-terminal C-terminal N-terminal 24

25 20 NMR conformers of PE00019A C-terminal N-terminal C-terminal N-terminal C-terminal N-terminal C-terminal N-terminal 25

26 26 Stereo View of side chain

27 Structure Homologues λ bacteriophage S. Typhimrium 27

28 28 Binding with Mg ++ metal

29 29 Conclusion NP_ is the first representative of unknown family Structure of NP_ was deduced without X-ray coordinates The NMR structure shows following features Two α-helix and two β-sheets A disorder region of 15 amino acid in between the sequence Binding experiment with Mg ++ metal indicated that protein does not oligomerized upon addition even 200 mM MgCl 2

30 30 NMR Structure of Protein YP_ , From Klebsiella pneumoniae Genome

31 YP_ is the first structural representative of the domain of unknown function DUF3315 (PF11776), Consists of 283 sequences from 112 different species. The 9.4 kDa polypeptide YP_ was selected with emphasis on members of Pfam families with no structure representative. Isolated from Klebsiella pneumoniae, a Gram-negative bacterium, a pathogen causing nosocomial pneumonia in immunocompromised patients as well as urinary tract infections (UTI), septicemia, and liver abscesses. Introduction

32 YP_ Number of amino acid: 83 GAAGIDQYAL KEFTADFTQF HIGDTVPAMY LTPEYNIKQW QQRNLPAPDA GSHWTYMGGN YVLITDTEGK ILKVYDGEIF YHR ω 1 ( 15 N) ppm ω 2 ( 1 H) ppm

33 33 Experiments: 4D-HACANH 5D-HACACONH 5D-CBCACONH 13 C-resolved NOESYs (ali & aro) 15 N-resolved NOESY 82.2% backbone assignments 89.2% Side chain assignments YP_

34 Statistics GS13720A

35 Validation Table

36 Secondary Structure Elements CA-CB (ppm) Sequence

37 C-terminal N-terminal

38 Ribbon representation of the conformer closest to the mean coordinates.

39 39 2D [ 15 N, 1 H]-HSQC spectrum of a 1.4 mM solution of uniformly 15 N-labeled YP_ recorded at 600 MHz and 298 K. Cross sections along ω 2 ( 1 H) through the cross peaks

40 No: Chain Z rmsd lali nres %id PDB Description 1: 2qzb-B PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY;2qzb-BPDB 2: 2qzb-A PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY;2qzb-APDB 3: 1su3-A PDB MOLECULE: INTERSTITIAL COLLAGENASE;1su3-APDB 4: 3kvp-D PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC;3kvp-DPDB 5: 3kvp-C PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC;3kvp-CPDB 6: 1gxd-B PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE;1gxd-BPDB 7: 1wmi-A PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013;1wmi-APDB 8: 1wmi-C PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013;1wmi-CPDB 9: 3ba0-A PDB MOLECULE: MACROPHAGE METALLOELASTASE;3ba0-APDB 10: 1rtg-A PDB MOLECULE: HUMAN GELATINASE A;1rtg-APDB 11: 1fbl-A PDB MOLECULE: FIBROBLAST (INTERSTITIAL) COLLAGENASE (MMP-1);1fbl-APDB 12: 3bpq-D PDB MOLECULE: RELB;3bpq-DPDB 13: 3bpq-B PDB MOLECULE: RELB;3bpq-BPDB 14: 1su3-B PDB MOLECULE: INTERSTITIAL COLLAGENASE;1su3-BPDB 15: 2clt-B PDB MOLECULE: INTERSTITIAL COLLAGENASE;2clt-BPDB 16: 2clt-A PDB MOLECULE: INTERSTITIAL COLLAGENASE;2clt-APDB 17: 2jxy-A PDB MOLECULE: MACROPHAGE METALLOELASTASE;2jxy-APDB 18: 1pex-A PDB MOLECULE: COLLAGENASE-3;1pex-APDB 19: 1gxd-A PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE;1gxd-APDB Structure Homologues DALI output

41 We have determine the structure determination of YP_ from Klebsiella pneumoniae in phosphate buffer at pH 6.0 using automated NMR protocol. YP_ exhibited a new structure fold and is the first representative of a new Pfam family of unknown function DUF3315 (PF11776). The protein showed a well-define globular structure comprises an anti-parallel β-sheet, an anti-parallel β- hairpin which is located perpendicularly to the β-sheet and five helices which surround the core of the protein. Conclusion

42 Saturation Transfer Difference (STD) NMR Spectroscopy 42

43 Saturation-Transfer-Difference (STD) NMR Group Epitope Mapping Saturation-Transfer-Difference (STD) NMR Group Epitope Mapping Reference spectrum STD Selective protein saturation

44 STD For Epitope Mapping and Binding Studies

45 Solubility High/ low affinity binding Specific and non specific bindings Limitation of STD NMR Spectroscopy

46 STD (Saturation Transfer Diffusion) Studies on α-Glucosidase Inhibitors

47 α-Glucosidase α-Glucosidase is present in the brush border membrane of the small intestine. It catalyzes the final step of carbohydrate digestion so that its inhibition suppresses the release of glucose from dietary origin The catalytic role of α-glucosidase makes it a therapeutic target to treat carbohydrate mediated diseases

48 Saccharomyces cerevisiae α -glucosidase (modeled) with Maltose as substrate in active site (Protein Model Portal). Saccharomyces cerevisiae iso-maltase (PDB-3AJ7) used for modeling with Maltose as substrate in active site. Acarbose – AGI, in active site of modeled Saccharomyces cerevisiae α - glucosidase (Guerreiro et al, 2013). Structural Features of Enzyme 48

49 α-Glucosidase Inhibition in Diabetes Acarbose as an example of AGI 49 (Arungarinathan et al., 2011)

50 Inhibitors of α-glucosidase delay the rate of conversion of disaccharide into monosaccharide. As a result, the postprandial blood glucose level is maintained at a lower level, leading to a decreased insulin demand. This approach is useful to manage glycemic index, independent to insulin in diabetic patients. Can be used as anti-obesity drugs Also have anti-viral drugs α-Glucosidase Inhibitors

51 How α-Glucosidase Inhibitors Work?

52 -GLUCOSIDASE INHIBITORY ACTIVITY α-Glucosidase (EC ) an exo type glycosylase that release α-glucoside from the non-reducing end side of the substrate. The aim of anti-diabetic therapy, both in insulin dependent diabetes mellitus and non-insulin dependent diabetes mellitus, is to achieve normoglycaemia (normal serum glucose level).

53 Mechanism of Action of α- Glucosidase Inhibitors Inhibition of the intestinal enzymes that break down the carbohydrates thus delay the absorption and digestion of carbohydrates in the gut Specifically target meal-related (postprandial) hyperglycemia, an independent risk factor for cardiovascular complications Control the glucose levels independently of insulin The effect on glycated hemoglobin (GHb) are comparable to metformin or thiazolidines

54 Mechanism of Action of α- Glucosidase Inhibitors α -Glucosidase inhibitors (AGI) as initial treatment for patients with Type 2 Diabetes Cause no hypoglycemic events Cause no weight gain Potential to be used as anti-obesity agents

55 Types of Inhibition 55 plus mixed-type inhibitor 1/V 1/V max app Mixed-type Competitive Non-competitiveUncompetitive

56 Acarbose H2OH2O DMSO H2OH2O CH 3 Sugar protons A B Protein irradiation point 56 (Ren et al., 2011) IC 50 ±SEM = 906±6.3 µM + Non-cytotoxic against 3T3 cell line

57 1-deoxynojirimycin H2OH2O DMSO H2OH2O A B Protein irradiation point 57 Competitive Inhibition IC 50 ±SEM = ±4.73 µM + Non-cytotoxic against 3T3 cell line

58 A B Reported Activities: Hypnotic Skin whitening Anticancer Antiangiogenesis Antioxidant

59 DMSO Protein irradiation point DMSO H2OH2O H2OH2O 224 H-1 H-4',5' I Impurities------I A B H-3' 59 Competitive Inhibition

60 DMSO Protein irradiation point DMSO H2OH2O H2OH2O 201 H-3',5' H-1 H-2',6' H-1 A B 60 Mixed-type Inhibition

61 Reported Activities: Antioxidant Alzheimer's disease Antibacterial Antimalarial Antidiabetic Antiparasitic

62 175 H2OH2O H2OH2O DMSO H-6'' H-2',6' H-3',5' H-4'' H-4 H-2',6' H-3',5' H-4 A B Protein irradiation point 62 Mixed-type Inhibition

63 176 H2OH2ODMSO H-6'' H-2',6' H-3'',4'' H-3',5' CH 2 CH 3 H2OH2O DMSO H-6'' H-3',5' A B Protein irradiation point 63 Non-Competitive Inhibition

64 184 – Non-inhibitor H2OH2O H2OH2O DMSO A B Protein irradiation point No STD Signals were observed 64 SolubilityHigh/Low affinity No interaction or no inhibition

65 A B Reported Activities: Antimicrobial Insecticidal Antioxidant Antibacterial Androgen Ligand Antidiabetic

66 259 DMSO H2OH2O B Protein irradiation point A B H-2',6' H-5'' H-3', 5' H-6'' H-2'' H-4 H-2',6' H-5'' H-3', 5' H-2'' H2OH2O DMSO 66 Mixed-type Inhibition

67 67 On Going Projects YP_ Thioredoxin protein YP_ Hypothetical phage protein YP_ Putative glycine cleavage H protein YP_ Putative membrane protein YP_ Putative membrane protein

68 68


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