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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, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
The Scripps Research Institute, La Jolla, CA, USA
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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 Structures X-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.
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The Steps inProtein 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 calculation5. Structure refinement6. Structure deposition
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Fig. 2 (2003) Progress in NMR Spectroscopy, 43, 105, Guntert.
The
AssignCalculateEvaluate
cycle
Automated NOE assignment
and structure calculation
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1D 1H-NMR screening1D 1H-NMR screening
Protocol for Automated NMR Structure Determination
1. NMR Sample 2. NMR Structure
Promising protein constructs and
solvent conditions
Promising protein constructs and
solvent conditions
2D [15N, 1H]-HSQC screening2D [15N, 1H]-HSQC screening
Structure quality protein solution NMR profile
Structure quality protein solution NMR profile
Automated backbone assignmentsAutomated backbone assignments
Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra
Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra
Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation
Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation
NMR structure solvedAccurate backbone fold
NMR structure solvedAccurate backbone fold
Interactive NMR structure refinement
Interactive NMR structure refinement
NMR structure refinedNMR structure refined
NMR structure validationNMR structure validationPDBPDB
No
7
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PJ03720C
TM0320
A. Folded globular protein
B. Non-globular proteinFolding
PG9814A
C. Aggregated or oligomerized protein
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[1H, 15N]-HSQC Spectra
TM0320GS13720A
PE00019A
9
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Number of expected peaks
Peak number (order with decreasing intensity)
Sig
nal
/No
ise
Signal-to-Noise NMR Profile
10
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Sig
nal
/No
ise
Peak number (order with decreasing intensity)
APSY quality
TM0320 NMR Profile
Number of expected peaks
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Peak Number (order with decreasing intensity)
PE00019A (A6)
0
50
100
150
200
250
300
350
400
450
500
550
600
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Sig
nal
/No
ise
88
Number of expected peaks
PE00019A NMR profile
Limited APSY quality
12
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NMR Experiments
3 APSY-NMR experiments20 35 20 projections each
3 APSY-NMR experiments20 35 20 projections each
3D-15N-resolved [1H, 1H]-NOESY3D-13C-resolved [1H, 1H]-NOESY (ali)3D-13C-resolved [1H, 1H]-NOESY (aro)
3D-15N-resolved [1H, 1H]-NOESY3D-13C-resolved [1H, 1H]-NOESY (ali)3D-13C-resolved [1H, 1H]-NOESY (aro)
Automated backbone assignmentAutomated backbone assignment
Interactive validation of backbone assignmentChemical shifts adaptation to NOESY spectra
Interactive validation of backbone assignmentChemical shifts adaptation to NOESY spectra
Automated [1H, 1H]-NOESY-based sidechain assignment, constraint
collection and structure calculation
Automated [1H, 1H]-NOESY-based sidechain assignment, constraint
collection and structure calculation
NMR structure solvedAccurate backbone
fold
NMR structure solvedAccurate backbone
fold
Interactive NMR structure refinementInteractive NMR structure refinement
NMR structure refinedNMR structure refined
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Software
GAPROGAPRO
MATCH
ASCAN
ATNOSCANDID
MATCH
ASCAN
ATNOSCANDID
UNIO
Automated backbone assignmentsAutomated backbone assignments
Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra
Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra
Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation
Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation
NMR structure solvedAccurate backbone fold
NMR structure solvedAccurate backbone fold
Interactive NMR structure refinement
Interactive NMR structure refinement
NMR structure refinedNMR structure refined
CYANA14
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Protein NP_888769.1,
• 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
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Number of amino acid: 141
Mol. Wt: 15.2 kDaExperiments:
4D-HACANH5D-HACACONH5D-CBCACONH
13C-resolved NOESYs (ali & aro)15N-resolved NOESY
75.6% backbone assignments
68.4% Side chain assignments
GMSQDLIRAAFEKRLSDWAKARTPALPVAWQNTKFTPPAAGVYLRAYVMPAATISRDAAGDHRQYRGVFQVNVVMPIGDGSRSAEQVAAELDALFPVNLVMQSGGLAVRVRTPISNGQPTTGDADHTVPISLGYDVQFYPE
16
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C-terminal
N-terminal
67
76
84
94
134
125
Sequence
CA
-CB
(p
pm
)
Secondary Structure Elements
19
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C-terminal
N-terminal
67
76
84
94
134
125
20
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Heteronuclear NOE Results
Sequence
A51
A124G78
Q31
N-terminalC-terminal
II
I
III IV
Rela
tive
in
ten
sity
22
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C-terminal
N-terminal
C-terminal
N-terminal
C-terminal
N-terminal
C-terminal
N-terminal
23
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C-terminalN-terminal
C-terminal
N-terminal
24
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20 NMR conformersof PE00019A
C-terminal
N-terminal
C-terminal
N-terminal
C-terminal
N-terminal
C-terminal
N-terminal
25
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Stereo View of side chain
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Structure Homologues
λ bacteriophage
S. Typhimrium
27
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Binding with Mg++ metal
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Conclusion
• NP_888769.1 is the first representative of unknown family
•Structure of NP_888769.1 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
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NMR Structure of Protein YP_001336205, From Klebsiella pneumoniae Genome
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• YP_001336205.1 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_001336205.1 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
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YP_001336205
Number of amino acid: 83
GAAGIDQYAL KEFTADFTQF HIGDTVPAMY LTPEYNIKQW QQRNLPAPDA
GSHWTYMGGN YVLITDTEGK ILKVYDGEIF YHR
10 20 30 40 50
60 70 80
ω1(15N)ppm
ω2(1H)ppm
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33
Experiments:
4D-HACANH5D-HACACONH5D-CBCACONH
13C-resolved NOESYs (ali & aro)15N-resolved NOESY
82.2% backbone assignments
89.2% Side chain assignments
YP_001336205
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Statistics GS13720A
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Validation Table
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Secondary Structure ElementsC
A-C
B (
pp
m)
Sequence
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C-terminal
N-terminal
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Ribbon representation of theconformer closest to themean coordinates.
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39
2D [15N,1H]-HSQC spectrum of a 1.4 mM solution of uniformly 15N-labeled YP_001336205.1 recorded at 600 MHz and 298 K.
Cross sections along ω2(1H) through the cross peaks
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No: Chain Z rmsd lali nres %id PDB Description 1: 2qzb-B 3.0 3.0 62 147 8 PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY; 2: 2qzb-A 2.9 3.0 58 145 9 PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY; 3: 1su3-A 2.3 5.1 49 415 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 4: 3kvp-D 2.3 2.4 40 49 13 PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC; 5: 3kvp-C 2.3 2.5 39 45 13 PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC; 6: 1gxd-B 2.3 3.0 42 623 12 PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE; 7: 1wmi-A 2.3 3.2 54 88 15 PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013; 8: 1wmi-C 2.3 3.1 54 88 15 PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013; 9: 3ba0-A 2.2 5.0 51 365 8 PDB MOLECULE: MACROPHAGE METALLOELASTASE; 10: 1rtg-A 2.2 3.6 46 203 13 PDB MOLECULE: HUMAN GELATINASE A; 11: 1fbl-A 2.1 5.1 49 367 8 PDB MOLECULE: FIBROBLAST (INTERSTITIAL) COLLAGENASE (MMP-1); 12: 3bpq-D 2.1 4.0 53 86 11 PDB MOLECULE: RELB; 13: 3bpq-B 2.1 4.0 56 85 16 PDB MOLECULE: RELB; 14: 1su3-B 2.1 5.1 48 416 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 15: 2clt-B 2.1 5.1 49 367 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 16: 2clt-A 2.1 4.9 49 367 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 17: 2jxy-A 2.1 4.9 50 194 8 PDB MOLECULE: MACROPHAGE METALLOELASTASE; 18: 1pex-A 2.1 4.8 48 192 10 PDB MOLECULE: COLLAGENASE-3; 19: 1gxd-A 2.1 3.5 45 624 13 PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE;
Structure HomologuesDALI output
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• We have determine the structure determination of YP_001336205.1 from Klebsiella pneumoniae in phosphate buffer at pH 6.0 using automated NMR protocol.
• YP_001336205.1 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.
Conclusion
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Saturation Transfer Difference (STD) NMR Spectroscopy
42
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Saturation-Transfer-Difference (STD) NMRSaturation-Transfer-Difference (STD) NMRGroup Epitope MappingGroup Epitope Mapping
Saturation-Transfer-Difference (STD) NMRSaturation-Transfer-Difference (STD) NMRGroup Epitope MappingGroup Epitope Mapping
Reference
spectrum
STDSTD
Selective
protein
saturation
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STD For Epitope Mapping and STD For Epitope Mapping and Binding StudiesBinding Studies
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Solubility
High/ low affinity binding
Specific and non specific bindings
Limitation of STD NMR Limitation of STD NMR SpectroscopySpectroscopy
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STD (Saturation Transfer Diffusion) STD (Saturation Transfer Diffusion) Studies on Studies on
α-Glucosidase Inhibitors α-Glucosidase Inhibitors
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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
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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 Structural Features of Features of
EnzymeEnzyme
48
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αα-Glucosidase Inhibition in Diabetes-Glucosidase Inhibition in DiabetesAcarbose as an example of AGI
49(Arungarinathan et al., 2011)
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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
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How α-Glucosidase Inhibitors Work?
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-GLUCOSIDASE INHIBITORY ACTIVITY
α-Glucosidase (EC 3.2.1.20) 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).
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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
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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
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(Z)
HO
OH
HO
HN
OH
O
O
O O
O
OH
HO
OH
HO
CH3
OH
HO
OH
OH
OH
Acarbose
H2O
DMSO
H2ODMSO
CH3
Sugar protons
Sugar protons
A
B
Protein
irradiation
poin
t
56
(Ren et al., 2011)
IC50±SEM = 906±6.3 µM + Non-cytotoxic against 3T3
cell line
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N
OH
OHHO
H OH
.HCl
1-deoxynojirimycin
H2O
DMSO
H2O
DMSO
A
B
Protein
irradiation
poin
t
57
Competitive Inhibition
Inhibitor (DNJ) concentrations
A: Line-weaver-Burk Plot of DNJ1/[S]
-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
1/V
-0.2
0
0.2
0.300 mM
0.200 mM
0.100 mM
0.025 mM
0.00 mM
IC50±SEM = 279.715±4.73 µM + Non-cytotoxic against 3T3
cell line
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A
B
Reported Activities:
HypnoticSkin whiteningAnticancerAntiangiogenesisAntioxidant
NH
NH
O
SO
R1
R2
R3
R4
R5
NH
NH
O
SO
R
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DMSO
Protein
irradiation
p
oint
DMSO
H2O
H2O
224
1' 2
7
NH6
5
NH 43
O
SO
O1
5'4'
3'2'
H-1
H-1
H-4',5'
H-4',5'
I-------Impurities------I
I-------Impurities------I
A
B
H-3'
H-3'
59
1/[S]-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
1/V
-0.2
0
0.2
Competitive Inhibition
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DMSO
Protein
irradiation
poin
t
DMSOH2O
H2O
201
1'
1
2
7
NH6
5
NH 43
O
SO
2'
3'
4'
5'
6'HO
H-3',5'
H-3',5'
H-1
H-2',6'
H-2',6' H-1
A
B
60
-10 0 10
1/V
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
Mixed-type Inhibition
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Reported Activities:
AntioxidantAlzheimer's diseaseAntibacterialAntimalarialAntidiabeticAntiparasitic
NH
N (E)
O
HO
R4
R1
R2
R3
R5R6
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175
5'
4'
3'
2'
1'
6'1
NH2
N3
4
(E)
O
1''
6''
5''
4''
3''
2''
HO
Br
HO
Br
H2O
H2O
DMSO
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
poin
t
62
1/[S]-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
1/V
-0.2
0
0.2
Mixed-type Inhibition
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176
5'
4'
3'
2'
1'
6'1
NH2
N3
4
(E)
O
1''
6''
5''
4''
3''
2''
HO
OH
HO
CH2CH3
H2O DMSOH-6''
H-2',6' H-3'',4''
H-3',5'
CH2
CH3
H2ODMSOH-6'' H-3',5'
A
B
Protein
irradiation
p
oint
63
1/[S]-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
1/V
-0.4
-0.2
0
0.2
0.4
Non-Competitive Inhibition
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5'
4'
3'
2'
1'
6'1
NH2
N3
4
(E)
O
1''
2''3''
4''
5''
6''
HO
OH
OEt
184 – Non-inhibitor
H2O
H2O
DMSO
DMSO
A
B
Protein
irradiation
p
oint
No STD Signals were No STD Signals were observedobserved
64
Solubility
High/Low
affinity
No interaction or no
inhibition
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A
B
Reported Activities:
AntimicrobialInsecticidalAntioxidantAntibacterialAndrogen LigandAntidiabetic
NH
N
O
N R5
R4
R3
R2
R1
NH
N
O
NR5
R4
R3
R2
R1
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259
DMSO
H2O
B
4'1
O
NH2
N3
CH4
1''
6''
5''
4''3''
2''
OH
OH
5'
6'
N1'
2'
3'
(Z)
Protein
irradiation
p
oint
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''
H2O
DMSO
66
1/[S]
-10 0 10
1/V
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
Mixed-type Inhibition
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67
On Going Projects
•YP_040532.1 Thioredoxin protein•YP_041423.1 Hypothetical phage protein•YP_039780.1 Putative glycine cleavage H protein•YP_041699.1 Putative membrane protein•YP_041210.1 Putative membrane protein
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68