Data analysis III: Biological NMR
Nuclear Magnetic Resonance is a unique and extremely powerful biophysical technique that
provides detailed information about individual atoms in biological macromolecules. NMR can
be used to solve a variety of biological and biomedical problems, including characterization
of:
–
Structure and dynamics for macromolecules (proteins and nucleic acids) including
different functional states and short-living intermediates in vitro and in vivo (inside the
living cells)
Functional conformational changes and flexibility of macromolecules
Allosteric pathways and mechanisms of signal transduction through protein structure
Intrinsically disordered proteins (IDP)
Molecular mechanisms of protein folding, misfolding and aggregations
Ligand and drug binding including characterisation of binding sites and molecular
contacts as well as thermodynamics and chemical kinetics of binding
Protein-protein and protein-nucleotide interactions including transient multicomponent
interactions
This project is primarily designed to introduce you to this state-of-the-art technique and
illustrate some practical aspects of modern protein NMR spectroscopy. Each section provides
a brief introduction to the topic and several examples. You will need to answer 2 NMR
questions as a part (50%) of Data analysis III: Biomolecular NMR spectroscopy and Mass
Spectrometry assessment that forms 25% of the overall module mark.
Other useful links:
Short intro to NMR:
Short intro in bioNMR from leading experts in the field:
Covid-19 NMR:
Where can I learn more about NMR (not required for this module)?
Lectures by James Keeler: http://www-keeler.ch.cam.ac.uk/lectures/
Lectures from James Keeler are also available on YouTube: https://youtu.be/nM7jQFhrvR0
Data analysis III: Biological NMR
Part 1: Prediction of protein secondary structure
Background information:
Protein secondary structure is the local spatial arrangement of a polypeptide’s backbone
atoms without regard to the conformations of its side chains. Protein secondary structure
prediction is often the first step toward tertiary structure prediction. It can also provide
information about protein stability, activity, and functions.
Backbone NMR chemical shifts can be used to accurately identify the location of secondary
structures in a protein. Positions of individual peaks in the NMR spectrum (i.e., chemical shifts)
are extremely sensitive characteristics of individual bonds as they depend on a local
microenvironment for a corresponding group of atoms and thus, provide information about
site-specific structural and dynamic features of proteins.
In folded proteins, different atoms have very different microenvironments. As a result, different
residues from a folded protein usually have very different chemical shifts. On the contrary, for
unfolded proteins variations of microenvironments are much smaller and chemical shifts only
depend on the local chemical environment, i.e., amino acid types of the residues itself and its
adjacent residues. These chemical shifts can be directly calculated from the protein sequence
using suggested random coil (fully unfolded) chemical shifts. In the folded protein, chemical
shifts strongly correlate with local protein structure. By comparison experimentally observed
chemical shifts to a set of residue-specific random coil (fully unfolded) chemical shifts, the
location and the type of protein structure can be identify using chemical shift index (CSI)
calculations.
This method assigns significant downfield secondary shifts (when the chemical shift is
significantly greater than the residue-specific random coil reference value) to ‘+1’s’, upfield
secondary shifts (when the chemical shift is significantly smaller than the residue-specific
random coil reference value) to ‘-1’s’ and small secondary shifts (when the chemical shift is
significantly is similar to the residue-specific random coil reference value) to ‘0’s’. A simple
graph showing CSI vs. residue numbers can be then generated. Any group of four or more -1
is assigned to an a-helix; any group of three or more +1 is assigned to a b-strand. All other
combinations are assigned as coil:
Data analysis III: Biological NMR
Question 1 (25 marks in total): Using the CSI method, predict secondary structure in the
nucleotide-binding domain (NBD) of bacterial Hsp70 (DnaK) using backbone NMR
assignments of the protein in the nucleotide-unbound state. Compare the CSI secondary
structure prediction with secondary structure predicted from the NBD amino acid sequence
using the PSIPRED algorithm.
Show and explain with the use of illustrations the results of this analysis; highlight the most
important differences in DnaK secondary structure predictions obtained using these two
approaches; briefly discuss key advantages and limitations of CSI and PSIPRED.
Instructions:
1) Download backbone chemical shifts for the protein from Biological Magnetic
Resonance Data Bank, BMRB, https://bmrb.io/ (BMRB accession code: 17209). Make
sure that you save an NMR-STAR v3 text file on your local computer. This file contains
information about the system, experimental conditions and backbone chemical shifts.
2) Use the CSI3.0 server (http://csi3.wishartlab.com/cgi-bin/index.php) to predict
secondary structure from backbone NMR chemical shifts of NBD. Upload the NMRSTAR v3 file from step 1 to the server and submit the job.
3) Use the PSRPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) to predict secondary
structure from the protein sequence (UniProt accession code P0A6Y8). Follow the
instructions on the website.
Note: You should only compare secondary structure for the nucleotide-binding domain of
DnaK (residues 1-388).
Presentation of your results: Show a graph(s) with secondary structure elements obtained
by CSI and PSPRED methods vs. protein residue numbers; highlight regions with the most
significant differences between two algorithms. You do not need to highlight and discuss each
individual change and each individual residue. Instead, you need clearly identify whether
different methods predict similar or different secondary structure elements for different parts
of the protein. You only need to compare the position of loops (unstructured regions), bstrands and a-helices. You can copy/paste and align graphs obtained from CSI and PSPRED
online tools or (if you prefer) make you own graph(s). Make sure that you provide a detailed
figure legend for each figure and cite all figures in the main text. Try to make your figures as
simple as possible while still providing a clear message.
The text for this answer (excluding figures) should NOT exceed 1 side of A4.
Data analysis III: Biological NMR
Part 2: Chemical shift perturbation (CSP) analysis
Background information:
The CSP analysis is an extremely sensitive tool to follow changes in protein structure and
dynamics upon any local or global conformational changes in a protein. Any local
perturbations (e.g., ligand binding, amino acid substitution, protonation of a side-chain,
conformational changes, etc.) within ~5-7 Å from the atom of interest usually result in chemical
shift perturbations. As a result, the CPS analysis is a very simple but extremely informative
experimental technique to study ligand binding, chemical reactions, pH titrations of functionally
important side chains, and characterization of allosteric (long-range) changes in the protein
structure.
To perform the CSP analysis, two or more 2D amide spectra
(HSQC) are usually recorded, for example:
(i) in the presence and in the absence of a ligand
OR
(ii) at different pH
OR
(iii) at different temperatures.
Any changes in peak positions in the NMR spectra can be
then monitored with high accuracy. Finally, significant CSPs
can be mapped into protein structure (if available) to identify
regions affected by a process (e.g., ligand binding, pH
changes, temperature).
Illustration from Prof Breeze’s
NMR lecture
Data analysis III: Biological NMR
Question 2 (25 marks in total): Hsp70 chaperones are key hubs in cellular proteostasis and
protein quality control networks in the cytoplasm and organelles. They facilitate protein folding,
disaggregation of misfolded proteins, protein degradation, protein refolding and assembly of
protein complexes. Hsp70 functions and activity rely on ATP binding to the Hsp70 N-terminal
nucleotide-binding domain (NBD) to allosterically alter substrate affinity to the C-terminal
substrate-binding domain (SBD). As the Hsp70 family is a putative drug target, initial screening
of the NBD of Hsp70 reveals a compound A that inhibits chaperone ATPase activity with Ki
of ca. 50 µM. To obtained molecular details of interactions between NBD and compound A,
2D amide spectra of 100 µM of 2H,15N labelled Hsp70 NBD were recorded in the absence and
present of 5 mM of the compound A. Using the chemical shift tables provided in
Question2_CSP.xlsx (Minerva), perform the chemical shift perturbation analysis of Hsp70
NBD; based on this analysis, suggest a binding site(s) and plausible mechanism(s) of action
for the compound A; suggest an independent experiment(s) to test your suggestions.
Instructions:
1. Using 1H and 15N chemical shifts recorded in the presence and absence of compound A
(Question2_CSP.xlsx), for each NBD residue calculate total chemical shift perturbations
(CPSs) using the following equation:
𝐶𝑆𝑃 = %Δ𝐶𝑆 (1𝐻)+ + [0.157Δ𝐶𝑆 (15𝑁)]+ ,
where ΔCS(1H) is the difference between proton chemical shifts recorded in the absence and
presence of compound A and ΔCS(15N) is the differences between nitrogen chemical shifts
recorded in the absence an presence of compound A.
2. Plot CSPs as a function of residue numbers and clearly identify residues with significant
(>0.2 ppm) CSPs. Attach this plot to your report. Make sure that you provide a detailed figure
legend and cite the figure in the main text. Try to make your figure as clear and simple as
possible.
3. Using PyMol, map residues with significant CSPs into 3D structure of NBD to identify the
binding site of the compound A (PDB accession code: 2KHO.pdb; the nucleotide-binding
domain corresponds to residues 1-388 in this structure). To identify the binding site and
suggest a plausible mechanism(s) of action, try to answer the following questions: Why only
some peaks are affected by binding to the compound A? Are all residues with significant CSPs
located close together? Are residues with significant CSPs located near functional sites of the
protein?
The text for this answer (excluding figures) should NOT exceed 1 side of A4.
Secondary*structure*prediction*(Question*1)
Secondary*structure*prediction*(Question*1)
We#can#compare#the#experimentally#observed#chemical#shifts#to#a#set#of#
residue7specific#‘random#coil’#chemical#shifts.#
ppm
‘+1’s’
‘&1’s’
49.11
53.56
55.01
59.56
Secondary*structure*prediction*(Question*1)
By#comparison#the#experimentally#observed#chemical#shifts#to#a#set#of#residue7
specific#‘random#coil’#chemical#shifts,#the#location#and#the#type#of#protein#structure#
can#be#identify#using#chemical(shift(index((CSI) calculations.#
Secondary*structure*prediction*(Question*1):
BMRB*data*base
https://bmrb.io/
17209
Secondary*structure*prediction*(Question*1):
BMRB*data*base
Save%this%file%on%your%local%computer
http://csi3.wishartlab.com/cgi3bin/index.php
Upload’the’17209’BMRB’file’
Secondary*structure*prediction*(Question*1):
CSI*output*(example)
Secondary*structure*prediction*(Question*1):
PSIPRED http://bioinf.cs.ucl.ac.uk/psipred/
UniProt accession,code:,P0A6Y8
https://www.uniprot.org/
Insert’the’protein’a.a.’sequence’
(residues’14388’for’NBD)
Secondary*structure*prediction*(Question*1):
CSI*vs.*PSIPRED
• Show&a&graph(s)&with&secondary&structure&elements&obtained&
by&CSI&and&PSIPRED&methods&vs.&protein&residue&numbers
• Highlight®ions&with&the&most&significant&differences&
between&two&algorithms
• Discuss
Chemical)shift)perturbation)analysis)(Question)2)
To perform the CSP analysis, two or more 2D amide
spectra (HSQC) are usually recorded, for example:
(i) in the presence and in the absence of a ligand
OR
(i) at different pH
OR
(i) at different temperatures.
Any changes in peak positions in the NMR spectra can be
then monitored with high accuracy. Finally, significant
CSPs can be mapped into protein structure (if available)
to identify regions affected by a process (e.g., ligand
binding, pH changes, temperature).
Chemical)shift)perturbation)analysis)(Question)2)
1.)Local)effect
The$site$of$perturbation
Unperturbed)protein
Perturbed)protein)
All)CSPs)within)517$Å from))
the)site)of)perturbations
Chemical)shift)perturbation)analysis)(Question)2)
2.)Long;range)effect
Long.range0effect
perturbation
Local0effect
CSPs)located)distant)(>5.70Å)0from))the)site)of)perturbations
Chemical)shift)perturbation)analysis)(Question)2):
Hsp70&binding&to&a&small&molecule&inhibitor
• Hsp70)chaperones)are)key)hubs)in)cellular)proteostasis
• Hsp70)functions)and)activity)rely)on)ATP)binding)to)the)Hsp70)ND
terminal)nucleotideDbinding)domain)(NBD))to)allosterically)alter)
substrate)affinity)to)the)CDterminal)substrateDbinding)domain)(SBD)
• As)the)Hsp70)family)is)a)putative)drug)target,)initial)screening)of)the)
NBD)of)Hsp70 reveals)a)compound(A that)inhibits)chaperone)ATPase)
activity)with)Ki of)ca.)50)µM
Chemical)shift)perturbation)analysis)(Question)2):)
Hsp70&binding&to&a&small&molecule&inhibitor
To obtained molecular details of interactions between NBD and
compound A, 2D amide spectra of 100 mM of 2H,15N labelled Hsp70
NBD were recorded in the absence and present of 5 mM of the
compound A.
You will perform the chemical shift perturbation analysis of Hsp70
NBD upon binding to the compound A.
Chemical)shift)perturbation)analysis)(Question)2):)
Hsp70&binding&to&a&small&molecule&inhibitor
!”# =
In#the#absence#of#compound#A
Δ!” 1′
(
+ 0.157Δ!” 15.
In#the#presence#of#compound#A
(
Chemical%shift%perturbation%analysis%(Question%2):%
Hsp70&binding&to&a&small&molecule&inhibitor
• Plot%CSPs%as%a%function%of%residue%numbers%and%clearly%identify%
residues%with%significant (>0.2%ppm)%CSPs
• Using%PyMol,%map%residues%with%significant%CSPs%into%3D%structure%of%
NBD%to%identify%the%binding%site%of%the%compound%A%
• Suggest%a%binding%site(s)%and%plausible%mechanism(s)%of%action%for%the%
compound%A
• Suggest%an%independent%experiment(s)%to%test%your%suggestions%
Software/web+servers:+
You$will$need$to$use$several$web$servers$for$data$analysis;$all$links$are$
provided$on$Minerva
PyMol
Additional)reading/References:)
• You$are$not expected$to$read$any$additional$literature$on$NMR$to$
answer$these$questions.$
• Instructions$on$data$analysis$servers$should$provide$your$all$necessary$
information$about$about$prediction$algorithms.$
• You[email protected]$references$maximum to$support$your$
statements/conclusions$about$biological systems.$$
Professionalism
You$should$aim$to$produce$professional$looking$documents
Use$illustrations$to$explain$your$key$points!
Make$sure$that$you$provide$detailed$figure$legends$and$cite$the$figures$
in$the$main$text.$Try$to$make$your$figures$as$clear$as$possible.
BUT$
Do$not$write$long$essay$answers!$Maximum$1$side$of$A4$for$each$
question,$excluding$figures.$
How$to$produce$a$good$figure?
• Good$protein$figures$
highlights$the$most$
important$specific$
regions$(residues)$in$the$
protein$(focus)
• They$are$colored only$so$
much$as$to$help$clarity
Figure$1:$Histogram$showing$the$backbone$amide$chemical$shift$
perturbations$observed$in$L94G.$The$changes$in$chemical$shift$
have$been$plotted$as$a$function$of$residue$number.$Dotted$line$in$
the$figure$represents$1SD.$
• The$figure$legend$
describes$the$figure$and$
contains$the$minimum$
information$needed$to$
interpret$the$message.$
How$to$produce$a$good$protein$figure?
• Good)protein)figures)
show)a)specific)region)in)
the)molecule)(focus)
• They)are)colored only)so)
much)as)to)help)clarity
Figure 1.)(A))Overview)of)AA)(yellow))bound)to)COX)sites)of)trCOX;2)(gray).)
(B))The)binding)pocket)(COX)site))and)the)hydrophobic)groove)of)trCOX;2)
with)AA.)The)key)residues)are)shown)in)green)(BIOC2303)example).
• The)figure)legend)
describes)the)figure)and)
contains)the)minimum)
information)needed)to)
interpret)the)message.)
Residue number
3
4
5
6
7
8
9
15
16
17
18
19
20
21
22
23
25
26
27
28
29
30
31
32
33
34
35
36
38
39
40
41
42
43
45
46
47
48
49
50
51
52
54
55
56
a.a. type
LYS
ILE
ILE
GLY
ILE
ASP
LEU
CYS
VAL
ALA
ILE
MET
ASP
GLY
THR
THR
ARG
VAL
LEU
GLU
ASN
ALA
GLU
GLY
ASP
ARG
THR
THR
SER
ILE
ILE
ALA
TYR
THR
ASP
GLY
GLU
THR
LEU
VAL
GLY
GLN
ALA
LYS
ARG
CS (1H) CS (15N)
9,009
122,386
8,06
122,792
7,553
120,381
9,046
109,874
8,525
123,554
8,127
124,419
6,595
129,16
8,509
124,145
9,07
127,573
8,991
128,402
8,474
113,504
8,425
121,368
8,361
128,511
8,233
114,442
8,484
117,882
7,659
119,351
9,153
124,895
8,334
128,31
7,928
126,017
8,094
120,445
8,058
118,671
8,404
121,702
7,645
116,574
8,142
108,5
7,814
119,831
9,096
123,705
7,413
109,111
9,173
117,863
9,22
125,076
8,399
127,715
8,607
128,949
9,367
127,552
7,833
121,465
8,634
114,984
7,871
116,515
7,664
107,509
7,557
121,003
8,549
120,659
9,141
127,427
8,54
120,77
8,665
114,141
10,523
129,34
6,437
116,763
8,152
122,408
8,02
115,403
57
58
60
61
63
64
65
66
67
68
69
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
91
92
93
94
95
96
98
99
100
101
102
103
104
105
106
107
GLN
ALA
THR
ASN
GLN
ASN
THR
LEU
PHE
ALA
ILE
ARG
LEU
ILE
GLY
ARG
ARG
PHE
GLN
ASP
GLU
GLU
VAL
GLN
ARG
ASP
VAL
SER
ILE
MET
PHE
LYS
ILE
ILE
ALA
ALA
ASN
GLY
ASP
ALA
TRP
VAL
GLU
VAL
LYS
GLY
7,053
7,19
7,063
7,485
8,613
6,566
6,905
9,666
6,337
9,165
7,774
7,691
7,752
7,029
9,937
7,99
8,698
8,733
8,2
7,277
9,07
9,146
6,876
8,133
7,52
7,339
8,353
7,249
7,279
7,793
5,956
8,985
7,833
8,657
7,475
8,615
7,305
7,811
7,981
8,39
9,587
8,827
8,485
8,63
9,295
8,608
111,908
124,105
108,778
120,196
115,408
112,584
117,052
128,127
115,489
115,206
118,527
114,855
114,796
119,361
115,223
118,096
121,281
122,992
111,851
123,606
127,479
118,812
120,258
116,736
119,695
121,709
122,115
110,824
116,221
122,188
115,235
122,169
129,208
121,114
123,16
126,205
114,435
108,415
124,498
121,948
130,019
115,067
125,639
126,474
128,018
104,849
108
109
110
111
114
115
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
135
136
137
138
139
140
141
142
144
145
146
147
148
149
150
151
152
153
155
156
157
158
159
GLN
LYS
MET
ALA
GLN
ILE
ALA
GLU
VAL
LEU
LYS
LYS
MET
LYS
LYS
THR
ALA
GLU
ASP
TYR
LEU
GLY
GLU
VAL
THR
GLU
ALA
VAL
ILE
THR
VAL
ALA
TYR
PHE
ASN
ASP
ALA
GLN
ARG
GLN
ALA
LYS
ASP
ALA
GLY
ARG
8,053
8,599
9,379
7,552
7,326
7,246
8,135
7,562
6,927
7,518
8,89
7,141
8,119
8,372
8,112
7,974
7,54
8,616
9,01
7,292
8,652
8,52
7,263
9,898
9,077
7,801
8,206
8,811
7,349
7,784
7,839
10,146
6,175
7,273
9,219
8,401
8,134
8,667
8,537
8,822
8,123
8,064
8,737
7,71
7,575
8,117
121,77
125,06
123,85
126,138
113,805
119,496
122,415
115,759
119,547
119,008
120,553
121,254
122,155
121,257
120,091
116,409
122,962
120,847
122,08
120,282
117,832
107,921
116,668
125,799
114,126
123,258
119,348
122,364
124,717
116,767
108,874
125,166
107,106
125,195
125,126
117,989
125,023
120,369
122,117
120,814
121,318
123,648
121,774
122,506
104,102
124,532
160
161
162
163
164
165
166
167
168
169
170
171
174
175
176
177
181
182
183
184
185
186
187
188
189
190
191
192
193
194
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
ILE
ALA
GLY
LEU
GLU
VAL
LYS
ARG
ILE
ILE
ASN
GLU
ALA
ALA
ALA
LEU
LEU
ASP
LYS
GLY
THR
GLY
ASN
ARG
THR
ILE
ALA
VAL
TYR
ASP
ASP
ILE
SER
ILE
ILE
GLU
ILE
ASP
GLU
VAL
ASP
GLY
GLU
LYS
THR
PHE
8,465
7,426
7,822
8,159
8,863
8,407
8,878
7,104
8,237
7,613
8,44
9,102
7,856
8,204
7,651
8,088
7,916
9,772
7,202
7,882
8,1
8,435
8,201
8,458
9,759
9,438
8,378
9,125
8,631
8,335
8,799
7,98
7,999
8,827
9,203
9,31
9,276
8,123
8,597
8,662
9,262
8,492
7,734
8,561
8,693
8,388
124,19
120,78
106,582
120,035
124,353
129,349
130,349
113,905
125,019
120,123
119,96
123,638
123,878
119,304
119,712
118,928
118,018
115,602
119,292
109,63
114,079
112,915
120,499
123,042
122,022
124,285
125,101
122,988
134,146
129,886
130,529
119,046
120,457
126,015
126,649
126,998
130,241
127,905
121,801
127,19
129,481
104,332
121,709
123,537
117,394
120,914
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
234
235
236
237
238
240
241
242
243
245
246
247
248
249
250
251
252
253
254
255
257
258
259
260
262
263
264
265
266
267
268
GLU
VAL
LEU
ALA
THR
ASN
GLY
ASP
THR
HIS
LEU
GLY
GLY
GLU
ASP
SER
ARG
LEU
ILE
ASN
LEU
VAL
GLU
GLU
LYS
LYS
ASP
GLN
GLY
ILE
ASP
LEU
ARG
ASN
ASP
LEU
ALA
MET
GLN
LEU
LYS
GLU
ALA
ALA
GLU
LYS
8,897
8,917
9,163
7,52
8,515
9,148
10,256
8,658
8,772
8,978
6,787
8,737
9,109
9,091
8,141
8,322
7,184
7,109
8,805
8,253
8,152
8,51
8,329
8,608
8,12
7,823
7,705
8,179
7,74
6,189
8,897
9,051
8,056
7,173
7,876
7,68
7,551
8,149
7,738
8,757
8,092
7,295
8,133
8,966
7,735
7,408
125,223
126,373
129,686
116,909
111,674
123,724
113,242
119,607
120,316
119,917
123,046
109,632
109,036
120,183
116,958
115,065
118,08
120,561
121,226
116,349
119,687
118,53
122,308
121,613
120,54
120,835
117,282
113,508
108,696
118,811
128,035
128,084
115,446
114,254
122,276
119,788
125,189
114,639
122,138
122,627
121,183
118,789
122,44
123,237
120,465
118,679
269
270
271
275
276
277
278
279
280
281
282
283
285
286
287
288
289
291
292
294
295
296
297
298
299
300
301
302
303
304
305
306
307
309
310
311
312
313
314
315
316
317
318
321
322
323
ALA
LYS
ILE
SER
ALA
GLN
GLN
THR
ASP
VAL
ASN
LEU
TYR
ILE
THR
ALA
ASP
THR
GLY
LYS
HIS
MET
ASN
ILE
LYS
VAL
THR
ARG
ALA
LYS
LEU
GLU
SER
VAL
GLU
ASP
LEU
VAL
ASN
ARG
SER
ILE
GLU
LYS
VAL
ALA
8,35
7,868
7,411
7,417
8,215
8,686
7,489
8,505
7,961
9,274
8,338
8,821
11,988
7,973
7,355
8,371
8,025
8,521
7,764
8,125
8,462
8,097
9,305
8,427
8,545
9,027
7,798
8,721
8,415
7,99
7,936
8,719
7,65
7,31
7,658
8,755
7,081
7,378
8,493
7,849
7,383
7,002
7,368
7,472
7,321
8,074
121,97
116,946
120,78
113,401
126,061
114,984
115,117
116,348
124,133
127,134
128,015
124,961
126,929
121,082
112,796
124,499
122,724
109,413
111,788
119,022
120,146
123,61
129,795
125,447
130,156
127,398
116,324
123,969
120,597
120,836
121,438
116,893
113,692
106,275
126,674
115,245
121,933
117,639
121,358
122,354
115,654
119,579
119,468
117,589
120,27
122,516
324
325
327
328
329
330
331
332
333
334
335
336
337
338
340
341
342
344
345
346
348
349
350
351
352
354
355
357
358
359
362
363
364
365
366
373
374
375
376
377
378
379
380
381
382
383
LEU
GLN
ALA
GLY
LEU
SER
VAL
SER
ASP
ILE
ASP
ASP
VAL
ILE
VAL
GLY
GLY
THR
ARG
MET
MET
VAL
GLN
LYS
LYS
ALA
GLU
PHE
GLY
LYS
ARG
LYS
ASP
VAL
ASN
ILE
GLY
ALA
ALA
VAL
GLN
GLY
GLY
VAL
LEU
THR
7,681
7,656
7,856
7,874
7,931
9,191
8,66
7,626
7,701
7,066
8,966
7,152
8,746
8,649
9,271
7,406
8,931
7,918
7,053
6,897
9,177
6,99
7,198
8,406
7,857
8,165
8,427
8,117
8,226
7,429
8,899
8,447
9,127
6,988
8,443
8,112
8,311
8,024
8,125
8,551
8,189
8,29
8,102
7,493
7,443
7,696
115,679
119,389
120,4
109,274
119,433
116,778
119,299
113,899
120,343
119,052
128,378
115,107
121,942
121,392
125,13
110,126
114,057
108,136
117,228
119,062
117,269
118,282
116,95
118,984
120,435
123,237
119,348
112,308
109,756
116,476
121,807
117,917
118,032
118,953
126,952
116,989
107,583
124,167
122,387
122,85
120,095
107,939
108,77
118,563
121,147
113,754
384
385
386
387
388
GLY
ASP
VAL
LYS
ASP
8,08
8,172
7,926
8,332
7,86
111,199
121,655
121,425
126,96
128,081
Residue number
a.a. type
3 LYS
4 ILE
5 ILE
6 GLY
7 ILE
8 ASP
9 LEU
15 CYS
16 VAL
17 ALA
18 ILE
19 MET
20 ASP
21 GLY
22 THR
23 THR
25 ARG
26 VAL
27 LEU
28 GLU
29 ASN
30 ALA
31 GLU
32 GLY
33 ASP
34 ARG
35 THR
36 THR
38 SER
39 ILE
40 ILE
41 ALA
42 TYR
43 THR
45 ASP
46 GLY
47 GLU
48 THR
49 LEU
50 VAL
51 GLY
52 GLN
CS (1H)
CS (15N)
9,027
122,431
8,068
122,694
7,564
120,270
9,013
109,885
8,568
123,566
8,062
124,286
6,523
129,798
8,552
124,440
9,112
127,632
9,015
128,484
8,455
113,630
8,389
121,124
8,363
128,619
8,252
114,364
8,478
118,031
7,650
119,325
9,152
124,828
8,400
128,060
8,081
126,407
8,108
120,324
8,056
118,776
8,367
121,788
7,697
116,765
8,126
108,560
7,822
119,905
9,170
123,295
7,365
108,539
9,257
117,819
9,495
125,273
8,440
127,034
8,436
128,050
9,295
127,124
7,833
121,111
8,595
114,714
7,878
116,495
7,691
107,623
7,549
120,994
8,539
120,516
9,167
127,676
8,487
120,991
8,590
114,090
10,475
129,466
54
55
56
57
58
60
61
63
64
65
66
67
68
69
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
91
92
93
94
95
96
98
99
100
101
ALA
LYS
ARG
GLN
ALA
THR
ASN
GLN
ASN
THR
LEU
PHE
ALA
ILE
ARG
LEU
ILE
GLY
ARG
ARG
PHE
GLN
ASP
GLU
GLU
VAL
GLN
ARG
ASP
VAL
SER
ILE
MET
PHE
LYS
ILE
ILE
ALA
ALA
ASN
GLY
ASP
ALA
6,446
8,350
7,959
7,131
7,147
7,022
7,471
8,659
6,535
6,911
9,651
6,386
9,378
7,813
7,712
7,728
6,983
9,905
8,056
8,813
8,804
8,185
7,276
9,100
9,094
6,868
8,101
7,528
7,390
8,338
7,265
7,259
7,758
5,991
8,868
7,832
8,669
7,484
8,637
7,302
7,828
7,989
8,394
116,140
123,714
114,728
112,411
124,280
107,693
120,114
116,008
112,332
116,814
128,277
116,796
116,021
118,827
114,577
114,626
119,299
114,878
118,445
121,370
123,012
111,887
123,604
128,158
119,406
120,250
116,285
119,651
121,321
122,075
110,933
116,649
122,147
115,181
122,126
129,022
121,080
123,180
126,329
114,419
108,431
124,532
121,861
102
103
104
105
106
107
108
109
110
111
114
115
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
135
136
137
138
139
140
141
142
144
145
146
147
148
149
TRP
VAL
GLU
VAL
LYS
GLY
GLN
LYS
MET
ALA
GLN
ILE
ALA
GLU
VAL
LEU
LYS
LYS
MET
LYS
LYS
THR
ALA
GLU
ASP
TYR
LEU
GLY
GLU
VAL
THR
GLU
ALA
VAL
ILE
THR
VAL
ALA
TYR
PHE
ASN
ASP
ALA
9,586
8,766
8,419
8,603
9,432
8,606
8,045
8,587
9,362
7,558
7,359
7,263
8,147
7,511
6,906
7,617
8,857
7,096
8,078
8,362
8,048
7,963
7,554
8,618
9,014
7,294
8,659
8,519
7,265
9,893
9,075
7,840
8,215
8,801
7,364
7,791
7,807
10,199
6,261
7,260
9,162
8,397
8,117
129,973
114,352
125,037
126,127
128,328
104,798
121,797
125,133
123,675
126,057
113,874
119,415
122,227
115,715
119,708
119,154
120,483
121,266
121,960
121,151
120,126
116,526
123,092
120,749
122,142
120,236
117,801
108,045
116,704
125,683
114,037
123,294
119,510
122,317
124,743
116,551
108,635
125,173
107,206
125,334
125,121
117,870
125,112
150
151
152
153
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
174
175
176
177
181
182
183
184
185
186
187
188
189
190
191
192
193
194
201
202
203
204
GLN
ARG
GLN
ALA
LYS
ASP
ALA
GLY
ARG
ILE
ALA
GLY
LEU
GLU
VAL
LYS
ARG
ILE
ILE
ASN
GLU
ALA
ALA
ALA
LEU
LEU
ASP
LYS
GLY
THR
GLY
ASN
ARG
THR
ILE
ALA
VAL
TYR
ASP
ASP
ILE
SER
ILE
8,677
8,557
8,807
8,146
8,054
8,723
7,722
7,568
8,119
8,468
7,416
7,823
8,159
8,871
8,391
8,890
7,130
8,226
7,603
8,522
8,880
7,929
8,263
7,670
8,033
7,878
9,711
7,241
7,842
8,098
8,434
8,194
8,463
9,680
9,439
8,364
9,054
8,541
8,532
9,463
7,946
7,810
8,780
120,537
122,100
120,771
121,256
123,516
121,736
122,562
103,933
124,673
124,197
120,854
106,538
120,155
124,406
129,090
130,195
114,012
124,888
120,449
119,683
123,155
123,661
119,215
119,886
118,732
118,021
115,544
119,400
109,449
114,023
112,941
120,409
123,414
122,105
124,014
124,837
123,153
133,040
129,817
130,685
119,518
120,324
126,185
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
234
235
236
237
238
240
241
242
243
245
246
247
248
249
250
251
ILE
GLU
ILE
ASP
GLU
VAL
ASP
GLY
GLU
LYS
THR
PHE
GLU
VAL
LEU
ALA
THR
ASN
GLY
ASP
THR
HIS
LEU
GLY
GLY
GLU
ASP
SER
ARG
LEU
ILE
ASN
LEU
VAL
GLU
GLU
LYS
LYS
ASP
GLN
GLY
ILE
ASP
9,169
9,287
9,275
8,129
8,657
8,641
9,257
8,491
7,713
8,584
8,672
8,401
8,883
8,924
9,157
7,441
8,522
9,224
10,348
8,917
8,667
9,040
6,779
8,719
10,069
9,748
7,949
8,338
7,214
7,157
8,833
8,269
8,194
8,529
8,347
8,614
8,129
7,823
7,725
8,192
7,724
6,195
8,895
126,995
127,707
130,501
128,077
121,936
126,961
129,453
104,289
121,571
123,805
117,465
120,807
125,098
126,783
129,959
116,496
112,702
124,818
113,067
120,834
119,050
121,170
124,768
107,675
108,704
123,073
118,005
115,391
118,309
121,021
121,229
116,430
120,089
118,939
122,337
121,733
120,496
120,814
117,276
113,596
108,619
118,829
128,090
252
253
254
255
257
258
259
260
262
263
264
265
266
267
268
269
270
271
275
276
277
278
279
280
281
282
283
285
286
287
288
289
291
292
294
295
296
297
298
299
300
301
302
LEU
ARG
ASN
ASP
LEU
ALA
MET
GLN
LEU
LYS
GLU
ALA
ALA
GLU
LYS
ALA
LYS
ILE
SER
ALA
GLN
GLN
THR
ASP
VAL
ASN
LEU
TYR
ILE
THR
ALA
ASP
THR
GLY
LYS
HIS
MET
ASN
ILE
LYS
VAL
THR
ARG
8,894
8,029
7,178
7,857
7,717
7,554
8,159
7,775
8,779
8,128
7,338
8,135
9,224
7,352
7,441
8,121
8,111
7,846
7,786
8,244
8,723
7,497
8,534
7,949
9,247
8,394
8,838
12,013
7,965
7,362
8,391
8,028
8,520
7,764
8,122
8,448
8,078
9,305
8,433
8,541
9,057
7,770
8,744
127,891
114,918
114,303
122,268
119,802
125,222
114,661
122,140
122,646
121,208
118,832
121,502
122,430
118,820
117,377
122,478
118,616
120,348
113,441
126,126
116,205
115,229
116,508
123,598
126,692
127,988
124,789
127,098
120,836
112,876
124,489
122,621
109,404
111,846
119,011
120,253
123,658
129,836
125,350
130,134
127,179
116,190
123,898
303
304
305
306
307
309
310
311
312
313
314
315
316
317
318
321
322
323
324
325
327
328
329
330
331
332
333
334
335
336
337
338
340
341
342
344
345
346
348
349
350
351
352
ALA
LYS
LEU
GLU
SER
VAL
GLU
ASP
LEU
VAL
ASN
ARG
SER
ILE
GLU
LYS
VAL
ALA
LEU
GLN
ALA
GLY
LEU
SER
VAL
SER
ASP
ILE
ASP
ASP
VAL
ILE
VAL
GLY
GLY
THR
ARG
MET
MET
VAL
GLN
LYS
LYS
8,409
7,959
7,894
8,765
7,662
7,304
7,633
8,682
7,123
7,228
8,513
7,841
7,340
7,044
7,364
7,540
7,390
8,100
7,629
7,660
7,863
7,878
7,913
9,179
8,672
7,666
7,695
7,067
8,919
7,168
8,728
8,549
9,260
7,079
9,594
7,542
6,690
6,822
9,295
7,032
7,065
8,346
7,991
120,759
120,832
121,032
116,917
113,429
106,480
126,755
114,990
122,195
117,741
121,013
122,910
114,623
119,897
119,437
117,750
120,364
122,576
115,679
119,442
120,335
109,286
119,433
116,805
119,587
113,966
120,294
119,346
128,297
115,356
122,098
121,154
125,294
110,347
115,971
109,503
117,596
119,217
117,619
117,784
116,576
119,060
120,177
354
355
357
358
359
362
363
364
365
366
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
ALA
GLU
PHE
GLY
LYS
ARG
LYS
ASP
VAL
ASN
ILE
GLY
ALA
ALA
VAL
GLN
GLY
GLY
VAL
LEU
THR
GLY
ASP
VAL
LYS
ASP
8,072
8,342
8,099
8,190
7,426
8,953
8,540
9,034
6,958
8,460
8,151
8,341
8,035
8,095
8,596
8,156
8,280
8,057
7,471
7,468
7,699
8,079
8,163
7,905
8,337
7,872
122,974
119,085
112,332
109,791
116,357
121,726
118,289
117,996
118,716
127,068
117,285
107,811
124,359
122,448
122,757
119,958
107,849
108,622
118,454
121,328
113,590
111,114
121,602
121,369
126,893
128,109
Purchase answer to see full
attachment
Nuclear Magnetic Resonance is a unique and extremely powerful biophysical technique that
provides detailed information about individual atoms in biological macromolecules. NMR can
be used to solve a variety of biological and biomedical problems, including characterization
of:
–
Structure and dynamics for macromolecules (proteins and nucleic acids) including
different functional states and short-living intermediates in vitro and in vivo (inside the
living cells)
Functional conformational changes and flexibility of macromolecules
Allosteric pathways and mechanisms of signal transduction through protein structure
Intrinsically disordered proteins (IDP)
Molecular mechanisms of protein folding, misfolding and aggregations
Ligand and drug binding including characterisation of binding sites and molecular
contacts as well as thermodynamics and chemical kinetics of binding
Protein-protein and protein-nucleotide interactions including transient multicomponent
interactions
This project is primarily designed to introduce you to this state-of-the-art technique and
illustrate some practical aspects of modern protein NMR spectroscopy. Each section provides
a brief introduction to the topic and several examples. You will need to answer 2 NMR
questions as a part (50%) of Data analysis III: Biomolecular NMR spectroscopy and Mass
Spectrometry assessment that forms 25% of the overall module mark.
Other useful links:
Short intro to NMR:
Short intro in bioNMR from leading experts in the field:
Covid-19 NMR:
Where can I learn more about NMR (not required for this module)?
Lectures by James Keeler: http://www-keeler.ch.cam.ac.uk/lectures/
Lectures from James Keeler are also available on YouTube: https://youtu.be/nM7jQFhrvR0
Data analysis III: Biological NMR
Part 1: Prediction of protein secondary structure
Background information:
Protein secondary structure is the local spatial arrangement of a polypeptide’s backbone
atoms without regard to the conformations of its side chains. Protein secondary structure
prediction is often the first step toward tertiary structure prediction. It can also provide
information about protein stability, activity, and functions.
Backbone NMR chemical shifts can be used to accurately identify the location of secondary
structures in a protein. Positions of individual peaks in the NMR spectrum (i.e., chemical shifts)
are extremely sensitive characteristics of individual bonds as they depend on a local
microenvironment for a corresponding group of atoms and thus, provide information about
site-specific structural and dynamic features of proteins.
In folded proteins, different atoms have very different microenvironments. As a result, different
residues from a folded protein usually have very different chemical shifts. On the contrary, for
unfolded proteins variations of microenvironments are much smaller and chemical shifts only
depend on the local chemical environment, i.e., amino acid types of the residues itself and its
adjacent residues. These chemical shifts can be directly calculated from the protein sequence
using suggested random coil (fully unfolded) chemical shifts. In the folded protein, chemical
shifts strongly correlate with local protein structure. By comparison experimentally observed
chemical shifts to a set of residue-specific random coil (fully unfolded) chemical shifts, the
location and the type of protein structure can be identify using chemical shift index (CSI)
calculations.
This method assigns significant downfield secondary shifts (when the chemical shift is
significantly greater than the residue-specific random coil reference value) to ‘+1’s’, upfield
secondary shifts (when the chemical shift is significantly smaller than the residue-specific
random coil reference value) to ‘-1’s’ and small secondary shifts (when the chemical shift is
significantly is similar to the residue-specific random coil reference value) to ‘0’s’. A simple
graph showing CSI vs. residue numbers can be then generated. Any group of four or more -1
is assigned to an a-helix; any group of three or more +1 is assigned to a b-strand. All other
combinations are assigned as coil:
Data analysis III: Biological NMR
Question 1 (25 marks in total): Using the CSI method, predict secondary structure in the
nucleotide-binding domain (NBD) of bacterial Hsp70 (DnaK) using backbone NMR
assignments of the protein in the nucleotide-unbound state. Compare the CSI secondary
structure prediction with secondary structure predicted from the NBD amino acid sequence
using the PSIPRED algorithm.
Show and explain with the use of illustrations the results of this analysis; highlight the most
important differences in DnaK secondary structure predictions obtained using these two
approaches; briefly discuss key advantages and limitations of CSI and PSIPRED.
Instructions:
1) Download backbone chemical shifts for the protein from Biological Magnetic
Resonance Data Bank, BMRB, https://bmrb.io/ (BMRB accession code: 17209). Make
sure that you save an NMR-STAR v3 text file on your local computer. This file contains
information about the system, experimental conditions and backbone chemical shifts.
2) Use the CSI3.0 server (http://csi3.wishartlab.com/cgi-bin/index.php) to predict
secondary structure from backbone NMR chemical shifts of NBD. Upload the NMRSTAR v3 file from step 1 to the server and submit the job.
3) Use the PSRPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) to predict secondary
structure from the protein sequence (UniProt accession code P0A6Y8). Follow the
instructions on the website.
Note: You should only compare secondary structure for the nucleotide-binding domain of
DnaK (residues 1-388).
Presentation of your results: Show a graph(s) with secondary structure elements obtained
by CSI and PSPRED methods vs. protein residue numbers; highlight regions with the most
significant differences between two algorithms. You do not need to highlight and discuss each
individual change and each individual residue. Instead, you need clearly identify whether
different methods predict similar or different secondary structure elements for different parts
of the protein. You only need to compare the position of loops (unstructured regions), bstrands and a-helices. You can copy/paste and align graphs obtained from CSI and PSPRED
online tools or (if you prefer) make you own graph(s). Make sure that you provide a detailed
figure legend for each figure and cite all figures in the main text. Try to make your figures as
simple as possible while still providing a clear message.
The text for this answer (excluding figures) should NOT exceed 1 side of A4.
Data analysis III: Biological NMR
Part 2: Chemical shift perturbation (CSP) analysis
Background information:
The CSP analysis is an extremely sensitive tool to follow changes in protein structure and
dynamics upon any local or global conformational changes in a protein. Any local
perturbations (e.g., ligand binding, amino acid substitution, protonation of a side-chain,
conformational changes, etc.) within ~5-7 Å from the atom of interest usually result in chemical
shift perturbations. As a result, the CPS analysis is a very simple but extremely informative
experimental technique to study ligand binding, chemical reactions, pH titrations of functionally
important side chains, and characterization of allosteric (long-range) changes in the protein
structure.
To perform the CSP analysis, two or more 2D amide spectra
(HSQC) are usually recorded, for example:
(i) in the presence and in the absence of a ligand
OR
(ii) at different pH
OR
(iii) at different temperatures.
Any changes in peak positions in the NMR spectra can be
then monitored with high accuracy. Finally, significant CSPs
can be mapped into protein structure (if available) to identify
regions affected by a process (e.g., ligand binding, pH
changes, temperature).
Illustration from Prof Breeze’s
NMR lecture
Data analysis III: Biological NMR
Question 2 (25 marks in total): Hsp70 chaperones are key hubs in cellular proteostasis and
protein quality control networks in the cytoplasm and organelles. They facilitate protein folding,
disaggregation of misfolded proteins, protein degradation, protein refolding and assembly of
protein complexes. Hsp70 functions and activity rely on ATP binding to the Hsp70 N-terminal
nucleotide-binding domain (NBD) to allosterically alter substrate affinity to the C-terminal
substrate-binding domain (SBD). As the Hsp70 family is a putative drug target, initial screening
of the NBD of Hsp70 reveals a compound A that inhibits chaperone ATPase activity with Ki
of ca. 50 µM. To obtained molecular details of interactions between NBD and compound A,
2D amide spectra of 100 µM of 2H,15N labelled Hsp70 NBD were recorded in the absence and
present of 5 mM of the compound A. Using the chemical shift tables provided in
Question2_CSP.xlsx (Minerva), perform the chemical shift perturbation analysis of Hsp70
NBD; based on this analysis, suggest a binding site(s) and plausible mechanism(s) of action
for the compound A; suggest an independent experiment(s) to test your suggestions.
Instructions:
1. Using 1H and 15N chemical shifts recorded in the presence and absence of compound A
(Question2_CSP.xlsx), for each NBD residue calculate total chemical shift perturbations
(CPSs) using the following equation:
𝐶𝑆𝑃 = %Δ𝐶𝑆 (1𝐻)+ + [0.157Δ𝐶𝑆 (15𝑁)]+ ,
where ΔCS(1H) is the difference between proton chemical shifts recorded in the absence and
presence of compound A and ΔCS(15N) is the differences between nitrogen chemical shifts
recorded in the absence an presence of compound A.
2. Plot CSPs as a function of residue numbers and clearly identify residues with significant
(>0.2 ppm) CSPs. Attach this plot to your report. Make sure that you provide a detailed figure
legend and cite the figure in the main text. Try to make your figure as clear and simple as
possible.
3. Using PyMol, map residues with significant CSPs into 3D structure of NBD to identify the
binding site of the compound A (PDB accession code: 2KHO.pdb; the nucleotide-binding
domain corresponds to residues 1-388 in this structure). To identify the binding site and
suggest a plausible mechanism(s) of action, try to answer the following questions: Why only
some peaks are affected by binding to the compound A? Are all residues with significant CSPs
located close together? Are residues with significant CSPs located near functional sites of the
protein?
The text for this answer (excluding figures) should NOT exceed 1 side of A4.
Secondary*structure*prediction*(Question*1)
Secondary*structure*prediction*(Question*1)
We#can#compare#the#experimentally#observed#chemical#shifts#to#a#set#of#
residue7specific#‘random#coil’#chemical#shifts.#
ppm
‘+1’s’
‘&1’s’
49.11
53.56
55.01
59.56
Secondary*structure*prediction*(Question*1)
By#comparison#the#experimentally#observed#chemical#shifts#to#a#set#of#residue7
specific#‘random#coil’#chemical#shifts,#the#location#and#the#type#of#protein#structure#
can#be#identify#using#chemical(shift(index((CSI) calculations.#
Secondary*structure*prediction*(Question*1):
BMRB*data*base
https://bmrb.io/
17209
Secondary*structure*prediction*(Question*1):
BMRB*data*base
Save%this%file%on%your%local%computer
http://csi3.wishartlab.com/cgi3bin/index.php
Upload’the’17209’BMRB’file’
Secondary*structure*prediction*(Question*1):
CSI*output*(example)
Secondary*structure*prediction*(Question*1):
PSIPRED http://bioinf.cs.ucl.ac.uk/psipred/
UniProt accession,code:,P0A6Y8
https://www.uniprot.org/
Insert’the’protein’a.a.’sequence’
(residues’14388’for’NBD)
Secondary*structure*prediction*(Question*1):
CSI*vs.*PSIPRED
• Show&a&graph(s)&with&secondary&structure&elements&obtained&
by&CSI&and&PSIPRED&methods&vs.&protein&residue&numbers
• Highlight®ions&with&the&most&significant&differences&
between&two&algorithms
• Discuss
Chemical)shift)perturbation)analysis)(Question)2)
To perform the CSP analysis, two or more 2D amide
spectra (HSQC) are usually recorded, for example:
(i) in the presence and in the absence of a ligand
OR
(i) at different pH
OR
(i) at different temperatures.
Any changes in peak positions in the NMR spectra can be
then monitored with high accuracy. Finally, significant
CSPs can be mapped into protein structure (if available)
to identify regions affected by a process (e.g., ligand
binding, pH changes, temperature).
Chemical)shift)perturbation)analysis)(Question)2)
1.)Local)effect
The$site$of$perturbation
Unperturbed)protein
Perturbed)protein)
All)CSPs)within)517$Å from))
the)site)of)perturbations
Chemical)shift)perturbation)analysis)(Question)2)
2.)Long;range)effect
Long.range0effect
perturbation
Local0effect
CSPs)located)distant)(>5.70Å)0from))the)site)of)perturbations
Chemical)shift)perturbation)analysis)(Question)2):
Hsp70&binding&to&a&small&molecule&inhibitor
• Hsp70)chaperones)are)key)hubs)in)cellular)proteostasis
• Hsp70)functions)and)activity)rely)on)ATP)binding)to)the)Hsp70)ND
terminal)nucleotideDbinding)domain)(NBD))to)allosterically)alter)
substrate)affinity)to)the)CDterminal)substrateDbinding)domain)(SBD)
• As)the)Hsp70)family)is)a)putative)drug)target,)initial)screening)of)the)
NBD)of)Hsp70 reveals)a)compound(A that)inhibits)chaperone)ATPase)
activity)with)Ki of)ca.)50)µM
Chemical)shift)perturbation)analysis)(Question)2):)
Hsp70&binding&to&a&small&molecule&inhibitor
To obtained molecular details of interactions between NBD and
compound A, 2D amide spectra of 100 mM of 2H,15N labelled Hsp70
NBD were recorded in the absence and present of 5 mM of the
compound A.
You will perform the chemical shift perturbation analysis of Hsp70
NBD upon binding to the compound A.
Chemical)shift)perturbation)analysis)(Question)2):)
Hsp70&binding&to&a&small&molecule&inhibitor
!”# =
In#the#absence#of#compound#A
Δ!” 1′
(
+ 0.157Δ!” 15.
In#the#presence#of#compound#A
(
Chemical%shift%perturbation%analysis%(Question%2):%
Hsp70&binding&to&a&small&molecule&inhibitor
• Plot%CSPs%as%a%function%of%residue%numbers%and%clearly%identify%
residues%with%significant (>0.2%ppm)%CSPs
• Using%PyMol,%map%residues%with%significant%CSPs%into%3D%structure%of%
NBD%to%identify%the%binding%site%of%the%compound%A%
• Suggest%a%binding%site(s)%and%plausible%mechanism(s)%of%action%for%the%
compound%A
• Suggest%an%independent%experiment(s)%to%test%your%suggestions%
Software/web+servers:+
You$will$need$to$use$several$web$servers$for$data$analysis;$all$links$are$
provided$on$Minerva
PyMol
Additional)reading/References:)
• You$are$not expected$to$read$any$additional$literature$on$NMR$to$
answer$these$questions.$
• Instructions$on$data$analysis$servers$should$provide$your$all$necessary$
information$about$about$prediction$algorithms.$
• You[email protected]$references$maximum to$support$your$
statements/conclusions$about$biological systems.$$
Professionalism
You$should$aim$to$produce$professional$looking$documents
Use$illustrations$to$explain$your$key$points!
Make$sure$that$you$provide$detailed$figure$legends$and$cite$the$figures$
in$the$main$text.$Try$to$make$your$figures$as$clear$as$possible.
BUT$
Do$not$write$long$essay$answers!$Maximum$1$side$of$A4$for$each$
question,$excluding$figures.$
How$to$produce$a$good$figure?
• Good$protein$figures$
highlights$the$most$
important$specific$
regions$(residues)$in$the$
protein$(focus)
• They$are$colored only$so$
much$as$to$help$clarity
Figure$1:$Histogram$showing$the$backbone$amide$chemical$shift$
perturbations$observed$in$L94G.$The$changes$in$chemical$shift$
have$been$plotted$as$a$function$of$residue$number.$Dotted$line$in$
the$figure$represents$1SD.$
• The$figure$legend$
describes$the$figure$and$
contains$the$minimum$
information$needed$to$
interpret$the$message.$
How$to$produce$a$good$protein$figure?
• Good)protein)figures)
show)a)specific)region)in)
the)molecule)(focus)
• They)are)colored only)so)
much)as)to)help)clarity
Figure 1.)(A))Overview)of)AA)(yellow))bound)to)COX)sites)of)trCOX;2)(gray).)
(B))The)binding)pocket)(COX)site))and)the)hydrophobic)groove)of)trCOX;2)
with)AA.)The)key)residues)are)shown)in)green)(BIOC2303)example).
• The)figure)legend)
describes)the)figure)and)
contains)the)minimum)
information)needed)to)
interpret)the)message.)
Residue number
3
4
5
6
7
8
9
15
16
17
18
19
20
21
22
23
25
26
27
28
29
30
31
32
33
34
35
36
38
39
40
41
42
43
45
46
47
48
49
50
51
52
54
55
56
a.a. type
LYS
ILE
ILE
GLY
ILE
ASP
LEU
CYS
VAL
ALA
ILE
MET
ASP
GLY
THR
THR
ARG
VAL
LEU
GLU
ASN
ALA
GLU
GLY
ASP
ARG
THR
THR
SER
ILE
ILE
ALA
TYR
THR
ASP
GLY
GLU
THR
LEU
VAL
GLY
GLN
ALA
LYS
ARG
CS (1H) CS (15N)
9,009
122,386
8,06
122,792
7,553
120,381
9,046
109,874
8,525
123,554
8,127
124,419
6,595
129,16
8,509
124,145
9,07
127,573
8,991
128,402
8,474
113,504
8,425
121,368
8,361
128,511
8,233
114,442
8,484
117,882
7,659
119,351
9,153
124,895
8,334
128,31
7,928
126,017
8,094
120,445
8,058
118,671
8,404
121,702
7,645
116,574
8,142
108,5
7,814
119,831
9,096
123,705
7,413
109,111
9,173
117,863
9,22
125,076
8,399
127,715
8,607
128,949
9,367
127,552
7,833
121,465
8,634
114,984
7,871
116,515
7,664
107,509
7,557
121,003
8,549
120,659
9,141
127,427
8,54
120,77
8,665
114,141
10,523
129,34
6,437
116,763
8,152
122,408
8,02
115,403
57
58
60
61
63
64
65
66
67
68
69
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
91
92
93
94
95
96
98
99
100
101
102
103
104
105
106
107
GLN
ALA
THR
ASN
GLN
ASN
THR
LEU
PHE
ALA
ILE
ARG
LEU
ILE
GLY
ARG
ARG
PHE
GLN
ASP
GLU
GLU
VAL
GLN
ARG
ASP
VAL
SER
ILE
MET
PHE
LYS
ILE
ILE
ALA
ALA
ASN
GLY
ASP
ALA
TRP
VAL
GLU
VAL
LYS
GLY
7,053
7,19
7,063
7,485
8,613
6,566
6,905
9,666
6,337
9,165
7,774
7,691
7,752
7,029
9,937
7,99
8,698
8,733
8,2
7,277
9,07
9,146
6,876
8,133
7,52
7,339
8,353
7,249
7,279
7,793
5,956
8,985
7,833
8,657
7,475
8,615
7,305
7,811
7,981
8,39
9,587
8,827
8,485
8,63
9,295
8,608
111,908
124,105
108,778
120,196
115,408
112,584
117,052
128,127
115,489
115,206
118,527
114,855
114,796
119,361
115,223
118,096
121,281
122,992
111,851
123,606
127,479
118,812
120,258
116,736
119,695
121,709
122,115
110,824
116,221
122,188
115,235
122,169
129,208
121,114
123,16
126,205
114,435
108,415
124,498
121,948
130,019
115,067
125,639
126,474
128,018
104,849
108
109
110
111
114
115
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
135
136
137
138
139
140
141
142
144
145
146
147
148
149
150
151
152
153
155
156
157
158
159
GLN
LYS
MET
ALA
GLN
ILE
ALA
GLU
VAL
LEU
LYS
LYS
MET
LYS
LYS
THR
ALA
GLU
ASP
TYR
LEU
GLY
GLU
VAL
THR
GLU
ALA
VAL
ILE
THR
VAL
ALA
TYR
PHE
ASN
ASP
ALA
GLN
ARG
GLN
ALA
LYS
ASP
ALA
GLY
ARG
8,053
8,599
9,379
7,552
7,326
7,246
8,135
7,562
6,927
7,518
8,89
7,141
8,119
8,372
8,112
7,974
7,54
8,616
9,01
7,292
8,652
8,52
7,263
9,898
9,077
7,801
8,206
8,811
7,349
7,784
7,839
10,146
6,175
7,273
9,219
8,401
8,134
8,667
8,537
8,822
8,123
8,064
8,737
7,71
7,575
8,117
121,77
125,06
123,85
126,138
113,805
119,496
122,415
115,759
119,547
119,008
120,553
121,254
122,155
121,257
120,091
116,409
122,962
120,847
122,08
120,282
117,832
107,921
116,668
125,799
114,126
123,258
119,348
122,364
124,717
116,767
108,874
125,166
107,106
125,195
125,126
117,989
125,023
120,369
122,117
120,814
121,318
123,648
121,774
122,506
104,102
124,532
160
161
162
163
164
165
166
167
168
169
170
171
174
175
176
177
181
182
183
184
185
186
187
188
189
190
191
192
193
194
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
ILE
ALA
GLY
LEU
GLU
VAL
LYS
ARG
ILE
ILE
ASN
GLU
ALA
ALA
ALA
LEU
LEU
ASP
LYS
GLY
THR
GLY
ASN
ARG
THR
ILE
ALA
VAL
TYR
ASP
ASP
ILE
SER
ILE
ILE
GLU
ILE
ASP
GLU
VAL
ASP
GLY
GLU
LYS
THR
PHE
8,465
7,426
7,822
8,159
8,863
8,407
8,878
7,104
8,237
7,613
8,44
9,102
7,856
8,204
7,651
8,088
7,916
9,772
7,202
7,882
8,1
8,435
8,201
8,458
9,759
9,438
8,378
9,125
8,631
8,335
8,799
7,98
7,999
8,827
9,203
9,31
9,276
8,123
8,597
8,662
9,262
8,492
7,734
8,561
8,693
8,388
124,19
120,78
106,582
120,035
124,353
129,349
130,349
113,905
125,019
120,123
119,96
123,638
123,878
119,304
119,712
118,928
118,018
115,602
119,292
109,63
114,079
112,915
120,499
123,042
122,022
124,285
125,101
122,988
134,146
129,886
130,529
119,046
120,457
126,015
126,649
126,998
130,241
127,905
121,801
127,19
129,481
104,332
121,709
123,537
117,394
120,914
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
234
235
236
237
238
240
241
242
243
245
246
247
248
249
250
251
252
253
254
255
257
258
259
260
262
263
264
265
266
267
268
GLU
VAL
LEU
ALA
THR
ASN
GLY
ASP
THR
HIS
LEU
GLY
GLY
GLU
ASP
SER
ARG
LEU
ILE
ASN
LEU
VAL
GLU
GLU
LYS
LYS
ASP
GLN
GLY
ILE
ASP
LEU
ARG
ASN
ASP
LEU
ALA
MET
GLN
LEU
LYS
GLU
ALA
ALA
GLU
LYS
8,897
8,917
9,163
7,52
8,515
9,148
10,256
8,658
8,772
8,978
6,787
8,737
9,109
9,091
8,141
8,322
7,184
7,109
8,805
8,253
8,152
8,51
8,329
8,608
8,12
7,823
7,705
8,179
7,74
6,189
8,897
9,051
8,056
7,173
7,876
7,68
7,551
8,149
7,738
8,757
8,092
7,295
8,133
8,966
7,735
7,408
125,223
126,373
129,686
116,909
111,674
123,724
113,242
119,607
120,316
119,917
123,046
109,632
109,036
120,183
116,958
115,065
118,08
120,561
121,226
116,349
119,687
118,53
122,308
121,613
120,54
120,835
117,282
113,508
108,696
118,811
128,035
128,084
115,446
114,254
122,276
119,788
125,189
114,639
122,138
122,627
121,183
118,789
122,44
123,237
120,465
118,679
269
270
271
275
276
277
278
279
280
281
282
283
285
286
287
288
289
291
292
294
295
296
297
298
299
300
301
302
303
304
305
306
307
309
310
311
312
313
314
315
316
317
318
321
322
323
ALA
LYS
ILE
SER
ALA
GLN
GLN
THR
ASP
VAL
ASN
LEU
TYR
ILE
THR
ALA
ASP
THR
GLY
LYS
HIS
MET
ASN
ILE
LYS
VAL
THR
ARG
ALA
LYS
LEU
GLU
SER
VAL
GLU
ASP
LEU
VAL
ASN
ARG
SER
ILE
GLU
LYS
VAL
ALA
8,35
7,868
7,411
7,417
8,215
8,686
7,489
8,505
7,961
9,274
8,338
8,821
11,988
7,973
7,355
8,371
8,025
8,521
7,764
8,125
8,462
8,097
9,305
8,427
8,545
9,027
7,798
8,721
8,415
7,99
7,936
8,719
7,65
7,31
7,658
8,755
7,081
7,378
8,493
7,849
7,383
7,002
7,368
7,472
7,321
8,074
121,97
116,946
120,78
113,401
126,061
114,984
115,117
116,348
124,133
127,134
128,015
124,961
126,929
121,082
112,796
124,499
122,724
109,413
111,788
119,022
120,146
123,61
129,795
125,447
130,156
127,398
116,324
123,969
120,597
120,836
121,438
116,893
113,692
106,275
126,674
115,245
121,933
117,639
121,358
122,354
115,654
119,579
119,468
117,589
120,27
122,516
324
325
327
328
329
330
331
332
333
334
335
336
337
338
340
341
342
344
345
346
348
349
350
351
352
354
355
357
358
359
362
363
364
365
366
373
374
375
376
377
378
379
380
381
382
383
LEU
GLN
ALA
GLY
LEU
SER
VAL
SER
ASP
ILE
ASP
ASP
VAL
ILE
VAL
GLY
GLY
THR
ARG
MET
MET
VAL
GLN
LYS
LYS
ALA
GLU
PHE
GLY
LYS
ARG
LYS
ASP
VAL
ASN
ILE
GLY
ALA
ALA
VAL
GLN
GLY
GLY
VAL
LEU
THR
7,681
7,656
7,856
7,874
7,931
9,191
8,66
7,626
7,701
7,066
8,966
7,152
8,746
8,649
9,271
7,406
8,931
7,918
7,053
6,897
9,177
6,99
7,198
8,406
7,857
8,165
8,427
8,117
8,226
7,429
8,899
8,447
9,127
6,988
8,443
8,112
8,311
8,024
8,125
8,551
8,189
8,29
8,102
7,493
7,443
7,696
115,679
119,389
120,4
109,274
119,433
116,778
119,299
113,899
120,343
119,052
128,378
115,107
121,942
121,392
125,13
110,126
114,057
108,136
117,228
119,062
117,269
118,282
116,95
118,984
120,435
123,237
119,348
112,308
109,756
116,476
121,807
117,917
118,032
118,953
126,952
116,989
107,583
124,167
122,387
122,85
120,095
107,939
108,77
118,563
121,147
113,754
384
385
386
387
388
GLY
ASP
VAL
LYS
ASP
8,08
8,172
7,926
8,332
7,86
111,199
121,655
121,425
126,96
128,081
Residue number
a.a. type
3 LYS
4 ILE
5 ILE
6 GLY
7 ILE
8 ASP
9 LEU
15 CYS
16 VAL
17 ALA
18 ILE
19 MET
20 ASP
21 GLY
22 THR
23 THR
25 ARG
26 VAL
27 LEU
28 GLU
29 ASN
30 ALA
31 GLU
32 GLY
33 ASP
34 ARG
35 THR
36 THR
38 SER
39 ILE
40 ILE
41 ALA
42 TYR
43 THR
45 ASP
46 GLY
47 GLU
48 THR
49 LEU
50 VAL
51 GLY
52 GLN
CS (1H)
CS (15N)
9,027
122,431
8,068
122,694
7,564
120,270
9,013
109,885
8,568
123,566
8,062
124,286
6,523
129,798
8,552
124,440
9,112
127,632
9,015
128,484
8,455
113,630
8,389
121,124
8,363
128,619
8,252
114,364
8,478
118,031
7,650
119,325
9,152
124,828
8,400
128,060
8,081
126,407
8,108
120,324
8,056
118,776
8,367
121,788
7,697
116,765
8,126
108,560
7,822
119,905
9,170
123,295
7,365
108,539
9,257
117,819
9,495
125,273
8,440
127,034
8,436
128,050
9,295
127,124
7,833
121,111
8,595
114,714
7,878
116,495
7,691
107,623
7,549
120,994
8,539
120,516
9,167
127,676
8,487
120,991
8,590
114,090
10,475
129,466
54
55
56
57
58
60
61
63
64
65
66
67
68
69
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
91
92
93
94
95
96
98
99
100
101
ALA
LYS
ARG
GLN
ALA
THR
ASN
GLN
ASN
THR
LEU
PHE
ALA
ILE
ARG
LEU
ILE
GLY
ARG
ARG
PHE
GLN
ASP
GLU
GLU
VAL
GLN
ARG
ASP
VAL
SER
ILE
MET
PHE
LYS
ILE
ILE
ALA
ALA
ASN
GLY
ASP
ALA
6,446
8,350
7,959
7,131
7,147
7,022
7,471
8,659
6,535
6,911
9,651
6,386
9,378
7,813
7,712
7,728
6,983
9,905
8,056
8,813
8,804
8,185
7,276
9,100
9,094
6,868
8,101
7,528
7,390
8,338
7,265
7,259
7,758
5,991
8,868
7,832
8,669
7,484
8,637
7,302
7,828
7,989
8,394
116,140
123,714
114,728
112,411
124,280
107,693
120,114
116,008
112,332
116,814
128,277
116,796
116,021
118,827
114,577
114,626
119,299
114,878
118,445
121,370
123,012
111,887
123,604
128,158
119,406
120,250
116,285
119,651
121,321
122,075
110,933
116,649
122,147
115,181
122,126
129,022
121,080
123,180
126,329
114,419
108,431
124,532
121,861
102
103
104
105
106
107
108
109
110
111
114
115
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
135
136
137
138
139
140
141
142
144
145
146
147
148
149
TRP
VAL
GLU
VAL
LYS
GLY
GLN
LYS
MET
ALA
GLN
ILE
ALA
GLU
VAL
LEU
LYS
LYS
MET
LYS
LYS
THR
ALA
GLU
ASP
TYR
LEU
GLY
GLU
VAL
THR
GLU
ALA
VAL
ILE
THR
VAL
ALA
TYR
PHE
ASN
ASP
ALA
9,586
8,766
8,419
8,603
9,432
8,606
8,045
8,587
9,362
7,558
7,359
7,263
8,147
7,511
6,906
7,617
8,857
7,096
8,078
8,362
8,048
7,963
7,554
8,618
9,014
7,294
8,659
8,519
7,265
9,893
9,075
7,840
8,215
8,801
7,364
7,791
7,807
10,199
6,261
7,260
9,162
8,397
8,117
129,973
114,352
125,037
126,127
128,328
104,798
121,797
125,133
123,675
126,057
113,874
119,415
122,227
115,715
119,708
119,154
120,483
121,266
121,960
121,151
120,126
116,526
123,092
120,749
122,142
120,236
117,801
108,045
116,704
125,683
114,037
123,294
119,510
122,317
124,743
116,551
108,635
125,173
107,206
125,334
125,121
117,870
125,112
150
151
152
153
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
174
175
176
177
181
182
183
184
185
186
187
188
189
190
191
192
193
194
201
202
203
204
GLN
ARG
GLN
ALA
LYS
ASP
ALA
GLY
ARG
ILE
ALA
GLY
LEU
GLU
VAL
LYS
ARG
ILE
ILE
ASN
GLU
ALA
ALA
ALA
LEU
LEU
ASP
LYS
GLY
THR
GLY
ASN
ARG
THR
ILE
ALA
VAL
TYR
ASP
ASP
ILE
SER
ILE
8,677
8,557
8,807
8,146
8,054
8,723
7,722
7,568
8,119
8,468
7,416
7,823
8,159
8,871
8,391
8,890
7,130
8,226
7,603
8,522
8,880
7,929
8,263
7,670
8,033
7,878
9,711
7,241
7,842
8,098
8,434
8,194
8,463
9,680
9,439
8,364
9,054
8,541
8,532
9,463
7,946
7,810
8,780
120,537
122,100
120,771
121,256
123,516
121,736
122,562
103,933
124,673
124,197
120,854
106,538
120,155
124,406
129,090
130,195
114,012
124,888
120,449
119,683
123,155
123,661
119,215
119,886
118,732
118,021
115,544
119,400
109,449
114,023
112,941
120,409
123,414
122,105
124,014
124,837
123,153
133,040
129,817
130,685
119,518
120,324
126,185
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
234
235
236
237
238
240
241
242
243
245
246
247
248
249
250
251
ILE
GLU
ILE
ASP
GLU
VAL
ASP
GLY
GLU
LYS
THR
PHE
GLU
VAL
LEU
ALA
THR
ASN
GLY
ASP
THR
HIS
LEU
GLY
GLY
GLU
ASP
SER
ARG
LEU
ILE
ASN
LEU
VAL
GLU
GLU
LYS
LYS
ASP
GLN
GLY
ILE
ASP
9,169
9,287
9,275
8,129
8,657
8,641
9,257
8,491
7,713
8,584
8,672
8,401
8,883
8,924
9,157
7,441
8,522
9,224
10,348
8,917
8,667
9,040
6,779
8,719
10,069
9,748
7,949
8,338
7,214
7,157
8,833
8,269
8,194
8,529
8,347
8,614
8,129
7,823
7,725
8,192
7,724
6,195
8,895
126,995
127,707
130,501
128,077
121,936
126,961
129,453
104,289
121,571
123,805
117,465
120,807
125,098
126,783
129,959
116,496
112,702
124,818
113,067
120,834
119,050
121,170
124,768
107,675
108,704
123,073
118,005
115,391
118,309
121,021
121,229
116,430
120,089
118,939
122,337
121,733
120,496
120,814
117,276
113,596
108,619
118,829
128,090
252
253
254
255
257
258
259
260
262
263
264
265
266
267
268
269
270
271
275
276
277
278
279
280
281
282
283
285
286
287
288
289
291
292
294
295
296
297
298
299
300
301
302
LEU
ARG
ASN
ASP
LEU
ALA
MET
GLN
LEU
LYS
GLU
ALA
ALA
GLU
LYS
ALA
LYS
ILE
SER
ALA
GLN
GLN
THR
ASP
VAL
ASN
LEU
TYR
ILE
THR
ALA
ASP
THR
GLY
LYS
HIS
MET
ASN
ILE
LYS
VAL
THR
ARG
8,894
8,029
7,178
7,857
7,717
7,554
8,159
7,775
8,779
8,128
7,338
8,135
9,224
7,352
7,441
8,121
8,111
7,846
7,786
8,244
8,723
7,497
8,534
7,949
9,247
8,394
8,838
12,013
7,965
7,362
8,391
8,028
8,520
7,764
8,122
8,448
8,078
9,305
8,433
8,541
9,057
7,770
8,744
127,891
114,918
114,303
122,268
119,802
125,222
114,661
122,140
122,646
121,208
118,832
121,502
122,430
118,820
117,377
122,478
118,616
120,348
113,441
126,126
116,205
115,229
116,508
123,598
126,692
127,988
124,789
127,098
120,836
112,876
124,489
122,621
109,404
111,846
119,011
120,253
123,658
129,836
125,350
130,134
127,179
116,190
123,898
303
304
305
306
307
309
310
311
312
313
314
315
316
317
318
321
322
323
324
325
327
328
329
330
331
332
333
334
335
336
337
338
340
341
342
344
345
346
348
349
350
351
352
ALA
LYS
LEU
GLU
SER
VAL
GLU
ASP
LEU
VAL
ASN
ARG
SER
ILE
GLU
LYS
VAL
ALA
LEU
GLN
ALA
GLY
LEU
SER
VAL
SER
ASP
ILE
ASP
ASP
VAL
ILE
VAL
GLY
GLY
THR
ARG
MET
MET
VAL
GLN
LYS
LYS
8,409
7,959
7,894
8,765
7,662
7,304
7,633
8,682
7,123
7,228
8,513
7,841
7,340
7,044
7,364
7,540
7,390
8,100
7,629
7,660
7,863
7,878
7,913
9,179
8,672
7,666
7,695
7,067
8,919
7,168
8,728
8,549
9,260
7,079
9,594
7,542
6,690
6,822
9,295
7,032
7,065
8,346
7,991
120,759
120,832
121,032
116,917
113,429
106,480
126,755
114,990
122,195
117,741
121,013
122,910
114,623
119,897
119,437
117,750
120,364
122,576
115,679
119,442
120,335
109,286
119,433
116,805
119,587
113,966
120,294
119,346
128,297
115,356
122,098
121,154
125,294
110,347
115,971
109,503
117,596
119,217
117,619
117,784
116,576
119,060
120,177
354
355
357
358
359
362
363
364
365
366
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
ALA
GLU
PHE
GLY
LYS
ARG
LYS
ASP
VAL
ASN
ILE
GLY
ALA
ALA
VAL
GLN
GLY
GLY
VAL
LEU
THR
GLY
ASP
VAL
LYS
ASP
8,072
8,342
8,099
8,190
7,426
8,953
8,540
9,034
6,958
8,460
8,151
8,341
8,035
8,095
8,596
8,156
8,280
8,057
7,471
7,468
7,699
8,079
8,163
7,905
8,337
7,872
122,974
119,085
112,332
109,791
116,357
121,726
118,289
117,996
118,716
127,068
117,285
107,811
124,359
122,448
122,757
119,958
107,849
108,622
118,454
121,328
113,590
111,114
121,602
121,369
126,893
128,109
Purchase answer to see full
attachment
