1. NMR Spectroscopy in Structural Biology
Atia-tul-Wahab, M. Iqbal Choudhary and Kurt Wüthrich,
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
X-Ray 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.
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.

3. Protein Structure Determination by NMR
The Steps in
Protein Structure Determination by NMR
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

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

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

6. Linking spin systems using nOe’s

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

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

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

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

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

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

13. NMR Experiments 3 APSY-NMR experiments 20 35 20 projections each
Automated backbone assignment
Interactive validation of backbone assignment
Chemical shifts adaptation to NOESY spectra
Automated [1H, 1H]-NOESY-based sidechain assignment, constraint collection and structure calculation
3D-15N-resolved [1H, 1H]-NOESY
3D-13C-resolved [1H, 1H]-NOESY (ali)
3D-13C-resolved [1H, 1H]-NOESY (aro)
NMR structure solved
Accurate backbone fold
Interactive NMR structure refinement
NMR structure refined

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

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
C-terminal 
N-terminal 
N-terminal 4 4      5  5  

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

17. Statistics

18. Validation Table

19. Secondary Structure Elements
C-terminal
134 67 
N-terminal 4 49 4 
CA-CB (ppm) 94  21 84 43 5 76  
125
Sequence

20. C-terminal 134 67  N-terminal 4 49 4  94  21 84 43 5 76  2  3 4 4 49 4  94  21 84 43 5 76  
125 5

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

22. Heteronuclear NOE Results
C-terminal
N-terminal 4 II 5 
A51 8
Relative intensity  6 
Q31  I  7
Sequence IV
III
A124
G78

23. C-terminal C-terminal  N-terminal  N-terminal 4 4     5  5         5 5    
N-terminal 4
N-terminal 4
C-terminal
C-terminal

24. C-terminal
C-terminal 5 5
N-terminal 4
N-terminal 4 8 8   6 6     7 7    

25. 20 NMR conformers of PE00019A C-terminal C-terminal N-terminal4 4  3 3 5    5  2 1 2 1 2 1 2 1   5 5   3
N-terminal 3
N-terminal 4 4
C-terminal
C-terminal

26. Stereo View of side chain

27. Structure Homologues
λ bacteriophage
S. Typhimrium

28. Binding with Mg++ metal

29. Conclusion NP_888769.1 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 MgCl2

30. NMR Structure of Protein YP_001336205,
From Klebsiella pneumoniae Genome

31. Introduction
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.

32. YP_001336205 GAAGIDQYAL KEFTADFTQF HIGDTVPAMY LTPEYNIKQW QQRNLPAPDA10 20 30 40 50
GAAGIDQYAL KEFTADFTQF HIGDTVPAMY LTPEYNIKQW QQRNLPAPDA
GSHWTYMGGN YVLITDTEGK ILKVYDGEIF YHR  60 70 80
Number of amino acid: 83
ω1(15N)
ppm
ω2(1H)
ppm

33. YP_001336205 4D-HACANH 5D-HACACONH 5D-CBCACONH
Experiments:
4D-HACANH
5D-HACACONH
5D-CBCACONH
13C-resolved NOESYs (ali & aro)
15N-resolved NOESY
82.2% backbone assignments
89.2% Side chain assignments

34. Statistics GS13720A

35. Validation Table

36. Secondary Structure Elements
CA-CB (ppm)
310 b  3
310
310 4
310 5 6 7
310
Sequence

37. 310 β1 β2
310
310
310 β3 β4 β5
310
310 5
310   3
310
310 4
310
N-terminal
310
C-terminal

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

39. 2D [15N,1H]-HSQC spectrum of a 1
2D [15N,1H]-HSQC spectrum of a 1.4 mM solution of uniformly 15N-labeled YP_ recorded at 600 MHz and 298 K.
Cross sections along ω2(1H) through the cross peaks

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

41. Conclusion
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 310-helices which surround the core of the protein.

42. Saturation Transfer Difference (STD) NMR Spectroscopy

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

44. STD For Epitope Mapping and Binding Studies44

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

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

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. Structural Features of Enzyme
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.
MAL12 binding site is composed of Asp214, Glu276, and Asp349 catalytic residues. In addition to the catalytic residues, molecular docking studies confirm Asp68, Tyr71, and Arg439 as important residues in the a-glucosidase inhibition
Guerreiro, L. R., Carreiro, E. P., Fernandes, L., Cardote, T. A., Moreira, R., Caldeira, A. T., Guedes, R. C., Burke, A. J. (2013). Five-membered iminocyclitol α-glucosidase inhibitors: synthetic, biological screening and in silico studies. Bioorganic and Medicinal Chemistry, 21(7):
Acarbose – AGI, in active site of modeled Saccharomyces cerevisiae α-glucosidase (Guerreiro et al, 2013).

49. α-Glucosidase Inhibition in Diabetes
Acarbose as an example of AGI
Type 1 : with damage to β pancreatic cells which in turn leads to insulin deficiency, thus increases the level of sugar in blood.
Type 2 : it is associated with resistance to insulin and insulin secretary defects causing relative deficiency. Obese, aged and people with less physical activity are prone to this type of diabetes. Women, hypertensive and dyslipidemic patients are at higher risk of NIDDM. Genetic factors also contribute to this type of diabetes but clear picture is not yet available.
Persistent hyperglycemia tends to glycate the proteins. These glycated proteins, in turn, start to accumulate the advance glycation endproducts (AGEs).
By far, the fda approved clinically used α-glucosidase inhibitors for niddm (voglibose, acarbose and miglitol) are competitive and reversible inhibitors of α-glucosidase. (Α-glucosidase inhibition in NIDDM)
Why we need better agi??
Tcurrent management with agi is associated with gastric side effects which includes flatulence, gastric cramps, diarrhea, etc. Another problem is frequent dosing i.E. AGI has to be taken with every meal. This also increases the cost of the therapy and patient compliance.
(Arungarinathan et al., 2011)

50. α-Glucosidase Inhibitors
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

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). 52

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 53

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 54

55. Types of Inhibition Mixed-type Competitive Non-competitive
plus mixed-type inhibitor
1/Vmaxapp
Mixed-type
Competitive
Comp inhi. Type of inhibition in which Km of the enzyme is not affected and inhibitor binds to active site.
Mixed type Type of inhibition in which inhibitor acts on both, Km and Vmax and inhibitor can bind to active site or any other site of enzyme.
Non comp Type of inhibition in which Vmax of the enzyme and inhibitor remains same and inhibitor binds to any other site, blocking the active site to perform action.
Un-comp Type of inhibition which does not allow product or substrate to leave active site as enzyme-substrate complex is inhibited. Kmapp may be lowered or increased relative to Km and Vmaxapp of enzyme is lowered.
Non-competitive
Uncompetitive

56. + Non-cytotoxic against 3T3 cell line A
Acarbose
IC50±SEM = 906±6.3 µM
+ Non-cytotoxic against 3T3 cell line A
Sugar protons
DMSO
CH3
H2O
Protein irradiation point B
The diagrammatic representations of the hydrogen bonds (dashed lines) and hydrophobic interactions (dashed-lined semicircles) formed by MGAM-C with inhibitor acarbose. NE2 of His1584 and OD2 of Asp1279 form hydrogen bonds with chemical groups OH Y=tyrosin w=trp F=phe R=Arg P=proline
Sugar protons
(Ren et al., 2011)
DMSO
H2O

57. Competitive Inhibition
1-deoxynojirimycin
IC50±SEM = ±4.73 µM
+ Non-cytotoxic against 3T3 cell line A
DMSO
H2O
Protein irradiation point B
DMSO
Competitive Inhibition
H2O

58. Thiobarbiturate Derivatives
Reported Activities:
Hypnotic
Skin whitening
Anticancer
Antiangiogenesis
Antioxidant B

59. Competitive Inhibition
224 A
DMSO
H-4',5'
I Impurities------I
H-1
H2O
H-3'
Protein irradiation point B
COMPOUND 224 HAD A FURAN RING INSTEAD OF BENZENE RING. THIS STRUCTURE HAD SHOWN RESULTS COMPARABLE WITH 205 I.E. THE FURAN IS LESS INTERACTIVE SINCE THE H-1', 3' AND H-4', 5' HAS REDUCED PEAK INTENSITY.
DMSO
H-1
Competitive Inhibition
H-4',5'
I Impurities------I
H2O
H-3'

60. Mixed-type Inhibition
201
Mixed-type Inhibition
H-2',6'
H-3',5'
H-1
H2O
DMSO A
Protein irradiation point B
MIXED TYPE OF INHIBITION IS REFERRED AS INTERACTION OF LIGAND WITH CATALYTIC POCKET AS WELL AS THE PROTEIN SURFACE. COMPOUND 201 AND 205 SHOWED THE SIMILAR PATTERN. H-1 PROTON, WHICH WAS SEEN INTERACTIVE IN ALL THE OTHER COMPOUNDS OF THIS SERIES, DEMONSTRATED LESSER (201) AND NO (205) INTERACTION WITH THE PROTEIN. IT CAN ALSO BE SEEN THAT BOTH THESE COMPOUNDS HAVE HYDROXYL GROUP ON BENZENE RING LIKE 199, WHICH MAY BE INTERACTING WITH THE WATER RESIDUES IN THE ACTIVE SITE TO STABILIZE THE LIGAND. HYDROGEN BONDING IS ALSO POSSIBLE WITH AVAILABLE HYDROPHOBIC OR HYDROPHILIC PARTNER IN THE ACTIVE POCKET. IT IS ALSO POSSIBLE THE CH OF TYROSINE PRESENT IN ACTIVE POCKET AS HYDROPHOBIC AMINO ACID, CH-Π INTERACTION IS TAKING PLACE BETWEEN THE BENZENE AND TYROSINE.
H-3',5'
DMSO
H-2',6'
H-1
H2O

61. Acylhydrazide Schiff Bases
Reported Activities:
Antioxidant
Alzheimer's disease
Antibacterial
Antimalarial
Antidiabetic
Antiparasitic

62. Mixed-type Inhibition
175
H-4
Mixed-type Inhibition A
H-6''
DMSO
H-2',6'
H-4''
H2O
H-3',5'
Protein irradiation point B
IT WAS OBSERVED THAT H-2', 6' AND H-3', 5' ARE INTERACTIVE PROTON PEAKS. FROM THIS OBSERVATION, WE CAN SAY THAT BENZENE RING NEAR TO CARBONYL END IS MORE INTERACTIVE AND IS MORE IMPORTANT FOR RENDERING THE ACTIVITY. IT IS ALSO OBSERVED THAT H-4'' AND H-6'' IS ALSO SHOWING WEAK INTERACTIONS. THUS WE CAN ASSUME THAT THE PART OF COMPOUND WHICH IS RESPONSIBLE FOR ACTIVITY IS PROBABLY THE BENZENE PART
DMSO
H-4
H-2',6'
H-3',5'
H2O

63. Non-Competitive Inhibition
176 A
H-3',5'
H-6''
CH3
H2O
DMSO
H-2',6'
CH2
H-3'',4''
Protein irradiation point B
THE INTERACTIVE PARTS OF THE LIGAND 176 ­ 178 WERE APPARENTLY FOUND TO BE BOTH THE BENZENE RINGS OF 176 ­ 178. FROM THIS REPEATED PATTERN IN 176 ­ 178, WE CAN ASSUME THAT PROBABLY THE PROTONS OF BENZENE RINGS ARE ACTING LIKE INTERACTIVE ANCHORS TO STABILIZE THE COMPOUND ON PROTEIN SURFACE TO GIVE THE ACTIVITY.
Non-Competitive Inhibition
H-6''
H-3',5'
DMSO
H2O

64. No interaction or no inhibition
184 – Non-inhibitor A
DMSO
H2O B
No STD Signals were observed
Protein irradiation point
No interaction or no inhibition
DMSO
Solubility
High/Low affinity
H2O

65. Nicotinic Schiff Bases
Reported Activities:
Antimicrobial
Insecticidal
Antioxidant
Antibacterial
Androgen Ligand
Antidiabetic B

66. Mixed-type Inhibition
259
DMSO A
H-2',6'
H-2''
H-4
Mixed-type Inhibition
H-6''
H-5''
H-3', 5'
H2O B B
. COMPOUND WAS FOUND TO MIXED TYPE OF INHIBITOR, INTERFERING TO THE FACT THAT IT HAS AN INTERACTION CAPABILITY WITH BOTH, CATALYTIC SITE AS WELL AS ENZYME SURFACE. TO IDENTIFY THE MOLECULE(S) RESPONSIBLE FOR INTERACTION, STD NMR SPECTROSCOPY WAS CARRIED OUT. IT WAS OBSERVED THAT PROTONS OF HETEROCYCLIC RING HAVING N AS ITS COMPONENT, WERE INTERACTING SINCE ALL THE PEAKS APPEARED IN THE DIFFERENCE SPECTRA. MOREOVER, PROTONS AROUND THE HYDROXYL FUNCTIONALITY WERE ALSO INTERACTING AS SEEN THROUGHOUT THE STUDY. THIS MAY SUGGEST THAT THE OH WHICH IS CAPABLE OF HYDROPHOBIC AS WELL AS HYDROPHILIC INTERACTION IS PLAYING ROLE IN CONTACT. THE CATALYTIC RESIDUES ASP AND GLU CAN BE STABILIZED BY SUCH OH GROUPS, MAKING THEM NON-INTERACTIVE WITH THE SUBSTRATE. THIS CAN BE THE POSSIBLE MECHANISM BY WHICH THIS PARTICULAR INHIBITOR IS ACTING.
Protein irradiation point
DMSO
H-3', 5'
H-2''
H-2',6'
H-5''
H2O

67. On Going Projects YP_040532.1 Thioredoxin protein
YP_ Hypothetical phage protein
YP_ Putative glycine cleavage H protein
YP_ Putative membrane protein
YP_ Putative membrane protein

68. Thank You