materials and methods sample preparation: five mg of the...
TRANSCRIPT
MATERIALS AND METHODS
Sample preparation: Five mg of the peptide was dissolved in 0.5 ml of a
90%H20, 10%O~ solution, and used as such in a 5mm tube for the NMR
experiments in water. Twenty one mg of deuterated Oodecylphosphotidylcholine
(d-OPC) (approximately 1:4 peptide:lipid ratio with lipid concentration above its
ernc) was added to the above solution and gently vortexed to allow the lipid to
dissolve into a homogenous solution, which was used in the NMR experiments to
determine the structure of the peptide induced by lipid.
To see if a higher concentration of d-OPC caused any change in the
conformation of the peptide (as proposed by McDonnell and Opella, 1993) a
second sample was prepared containing 2mg of peptide and 60 mg of OPC. As a
higher concentration would be detrimental to the NMR experiment,· resulting in
line broadening, the concentration of NKA was reduced. This sample is alluded to
as "NKA - high OPC" in the following discussion to distinguish it from the first
sample, henceforth called "NKA - low OPC".
Kassinin was prepared as in NKA-Iow OPC, 20 mg of d-OPC being added
to 5 mg of the peptide in water.
The p!-f was measured to confirm that it was acidic and left unadjusted.
Data Acquisition: All spectra for NKA-Iow OPC and Kassinin were measured on
a Bruker AMX-500 spectrometer. The spectra for NKA-high OPC were acquired
on a Varian UNITY 600 MHz spectrometer. Both instruments are located at the
National facility for high field NMR at the Tata Institute of Fundamental research,
Mumbai.
Experimental details are as follows:
All experiments were conducted with 2048 data points in the F1 dimension.
Shimming was performed on the deuterium lock signal, and an initial 90 0 pulse
determined using the water peak. Solvent suppression was achieved by
irradiation of the solvent frequency during recycle delay.
73
A TOCSY with mixing time 30ms and no trim pulses, OQFCOSY with 64
transients and 512 increments were collected. NOESY experiments 64 transients
and 350 increments were collected at mixing times of 150, 200, 250, 300 and 350
ms.
Temp was 298K in all cases except for the temperature coefficient
experiments where 1 Os were collected from 283 to 313K at increments 5°K. Data
points in F2 were 2K in all cases, Spectral width was 5050.50 Hz for the TOCSY
and 4629.63 Hz in the NOESY buildups, to reduce experiment time. In general,
care was taken to see that there were no folded peaks and the spectral width set.
As the baseline maybe distorted in purely absorptive spectra (TOCSY, NOESY
and ROESy), the spectral width was set such that there was sufficient space
between the terminal resonance signals and the edges of the spectral window.
Processing: All NMR spectra were processed on a Silicon Graphics R4000,
IRIS Indigo workstation using the computer program FELIX, (Molecular
simulation Inc., USA.)
Resolution enhancement was provided by using a sinebell apodisation
function shifted by 90°. The residual water peak can be removed by using a
deconvolution function, however since there is a dampening produced on the.
cross peaks close to the water peak form the tails of the deconvolution
function, the residual water peak was left untouched for spectra from which
peak intensities were measured. The deconvolution function also causes
severe distortion of the baseline with lipid samples - possibly as the water
signal contains some gaussian contributions as a result of the lipid surface.
The FlO was fourier transformed to generate the frequency spectrum and zero
and first order phase correction applied to convert out of phase spectra into
absorptive peaks (Fig.2.1 in results).
74
Measurement of coupling constants:
3 J H _a
and 3 Jaj\ can be measured from the COSY antiphase cross-
peaks between the amide and Ca protons of the side chain. FELIX has an
automated procedure to perform this function.
Peak picking and volume measurement.
Peak picking of NOESY spectra is initially performed using an
automated routine to select all peaks above a given threshold. The peak areas
determined by a square or rectangular box centred where the peak is at its
highest value. The peaks are then filtered by removing diagonal peaks and
non-symmetrical cross-peaks. Peak volumes are measured by FELIX by
calculating the volume adding the intensities of each point within the peak box.
After assigning the peptide (described in Results section of this chapter),
cross-peaks were assigned and verified by plotting the build-up of peak of
intensity vis mixing time. This allows inconsistent peaks (such as multiply
assigned overfapping cross peaks and those which are artefacts created by
noise, especially T1 noise) to be identified.
Restraint generation using Felix.
Using the initial linear region~of the build up curve, distance constraints
can be generated relative to a known distance a reference peak. The ~ cross
peaks of serine 5 and NH~ crosspeak of glycine-8 were used as reference
peaks, for NKA and for Kassinin. NOE distances were bracketed as weak o 0 0
(3.5-6.0A), medium (2.5-3.5 A) and strong (1.5-2.5 A). A fourth category -
o
qualitative, corresponding to (-1.0 - 6.0 A), was used to describe peaks which
were multiply assigned or were overfaps: For simulated annealing, the lower
distance constraint was set at -1°A, to allow for correct folding.
75
Structure generation.
We have used Distance Geometry (DG) and Simulated Annealing to
generate folded conformations for the peptides under study. The DIANA
package is used to demonstrate the use of DG, but detailed analysis is
hampered as visual ising the resultant conformations was not possible due to
incompatibility between the co-ordinate files of the program and Insight II
(MSI, San-Diego).
Distance Geometry
The 3-D structure was computed using the program DIANA and supporting
programs CAUBA, HABAS and GLOMSA (Guntert et aI., 1991). Diana makes
use of the target function method (Braun and Go, 1985) to gradually fit an initially
randomised starting structure to the conformational constraints collected with the
use of NMR experiments starting with intraresidual constraints only, and
increasing the target size stepwise to the length of the complete polypeptide
chain.
The supporting program CALIBA was used to calibrate the NOE peaks. A list
of assignments and NOE connectivities was output from FELIX, which was used
extensiveJy to analyse the data, and the format modified to use as input for
CALIBA. Calipration of the NOE peaks were made separately for the different
dasses of intraresidual, sequential medium-range and long-range backbone and . ,
long-range constants (Wuthrich, 1986). The list was processed and calibrated
into a series of upper distance constraints, which was used by HABAS, along
with the J~upling infomlation to determine stereospecific assignments and
generate angle constraints.
The REDAC strategy which provides partial feedback of structural information
from all conformers that were calculated upto a maximal level Lmax. Redundant
dihedral angle constraints are generated for all those residues found to be
acceptable in at least a predefined minimal number of conformers by taking the
two extreme dihedral angle values in the group of acceptable conformers as
upper and lower bounds.
76
Simulated Annealing
The simulated Annealing was performed using the molecular
mechanics and dynamics simulation program Discover® (Molecular
Simulations Inc., San Diego). A protocol developed by Nilges et aI., (1988)
was used in our experiments. The protocol is outlined in fig.2.D.
Fig. 2.E is a summary of the facilities available with Felix, that have
been used in the following analysis of neurokinin-A and Kassinin.
77
FELIX (NMR analysis software)
nD'NMRdata processing
frequency domain spectra
. Fourier Transform ~ LP
Window Functions
Analysis
measure ppm (used in assignments)
j-couplings (scalar couplings used in dihedral
STRUCTURE GENERATION
Fig. 2.E: NMR processing and analysis using FELIX.
78
2
point (x 1000)
... ------------------
2 point (x 1000)
Fig. 2.1 : Steps involved in processing tbe r.i.d. Panel on left from top to bottom shows i) the f.i.d. ii) after apodisation using as shifted sinebell function iii) after fourier transformation to generate the frequency spectrum and iv) after phase correction. Panel on right top shows the tid after treatment with a sine deconvolution function to remove the residual water signal. The remaining steps are as on the left. The sample above is ofNKA in DPC solution, and the deconvolution function to remove the residual water signal is not as effective as in water samples.
79
I "IP <XJ
• 0
Q
g . /) /)
(J
0
"i?()
0 0
(I <Ja~
I
B.O I
0 D
-oCt
.0 I
~ •
II
--=--- 0" cf' IJ
on 0
~7 0
II
/ tf~
00 <9 q
/) __ 00
I
6.0 4.0 D1 (ppm)
Fig. 2.2 TOCSY spectrum ofNKA in water,
(;> 0 ,~-v---
~C3
0 OJd
B arU t::>
(1~ .~ rr
0~ '0 (>.
rJ 0() 0
0
0 • () 0 .. 0" 00 0
0 g
en '10 <>0
0 0 . I li
I
2.0
The residual water signal has been removed by deconvolution during processmg.
0 f- •
C\I
0 ~
E' Q.. ~ ~
-0 C -<0
0 f-cti
80
, /
t
C
-I
I
B.O
•
I I
6.0 4.0
D1 (ppm)
Fig. 2.3 DQF-COSY spectrum ofNKA in water.
4 t - -..
I
2.0
Positive peaks are shown in cyan, negative in dark blue. The residual water signal has been removed by deconvolution during processing.
81
I I . . ~ 0 ::\ - 0 ~
.,,, · ". ~p!t~ ~ -0
• I) 0 '~ ~ ' , , >:0_: . c u,~ 1- -.
/ I
{ ; I
a <I ,
. ~ u ~ L
J ' :
1 . • 0
(> a -.. ~ u~ ~ 00 0" . . ~ 0 0 ~ J_-====---= " · ",.
- .-= ~:.... {/ ""-----~"'==- P '" "
/ t -
" I
~ i
.. .
. / o 0 · J r-. ~
.4 ". ~ on D ;
l I I I I
8.0 6.0 4.0 2.0
01 (ppm)
Fig. 2.4 ROESY spectrum (300 ms mixing time) ofNKA in water. Positive peaks are shown in black, negative peaks in red. Dipolar coupling causes negative cross peaks in 2D ROESY. The residual water signal was removed by deconvolution during processing.
0 "1-
E' Q. ~ "t-
O Q <0
82
Fig 2.5: Proton chemical shifts of Neurokinin A (NK-A) in water. Chemical shifts are relative to tetramethylsilane.
Proton chemical shifts (ppm)
Amino acid NH CHu CHP Others
His 4.345 3.407 8.697; 7.403 Lys 8.858 4.458 1.802 1.687; 1.422 Thr 8.494 4.361 4.223 1.225 Asp 8.545 4.693 2.761 Ser 8.225 4.400 3.782 Phe 8.301 4.669 3.14/3.059 7.255; 7.47; 7.30 Val 8.004 4.079 2.031 0.901 Gly 8.040 3.90 Leu 8.150 4.355 1.645 0.939; 0.893 Met 8.361 4.467 2.614 2.022; 2.102
83
I I I I I I I II
I I M1() I I L~ II V7
*2 I I I I I I II
I D~ I I I S~ I cr? CO rm I p6 1 I II K2
: I ~ , c:::i ff -~1 I I I I I ~ II
&' I I I I I I II
1 ~ II I I II ~ ' L ,r
' I 1 1 I I II
i fJ I I I I I II ' . 1
(i) I I I I I I II I \ ~ ~Q I I I I I I II (0
I I I I C I ~; 11 ck 6 111 ""-o (JJ I I I 1 13 1 I
J3 ~ I Ie I I I I I I I ' ~ I I I I I I II
I 1 J3~\1 I I II I I l I I I I ~ J3 ~ t\ I I E I I I II V I I I I I I II
I I I I I I II ~
( I I I I I I II I , C'\i E' II $1
I I II I I 1 I I I II Q..
I \ I I I II ~ ,<::1: I
, I I k I I I " Q ~
J3 I I I I a I I I I I I
I I
~t~ I
CV E f~ I I '< "" I I I I M I I I I I I
Ie I I I I I I
I I I I I
I I I I ,1 I I I I I I I I , ,(~ I 11 4 11 .' I I ' I . l • 'I:? II II d>~ a ~ C I I I I I I I
~ I I I I <;!#'a ~ J3 :
I II
I I I II
B.1 7.2
01 (ppm)
Fig. 2.6 Overlay of the fingerprint regions of TOCSY (black) and ROESY (negative levels in red). Only sequential cross peaks are visible. Vertical lines are drawn to show the amino acid spin-system, and meet the X-axis at the NH resonance.
84
o
o
o o
c;)
o o
03 0
D 0 8 o •
o , tfJ
o tI ' , eo' 0
9.0
rJ
o
(>0 0 00 ? 00 co
o cJl o
. i . ~ . . ' \
6.0 3.0
D1 (ppm)
Fig. 2.7: TOCSY spectrum of5 mg NKA with 20 mg DPC (1:4 NKA:DPC).
a tv)
a <ci
a 0)
Henceforth called NKA-Iow DPC (The residual water signal was removed by deconvolution during processing.)
85
E' 0.. -S ,.-. a
.,,~ ~.
• ,I ,. Q'\1
CI cD: . o •
t tr 8
0 ~ 0 e C
o .e • 0 -C\i 0 c.
0 ~O fIJ 0
o e ;. I-0 0 • ,(. /-.,
.. 0 •
~ G9~ 1; i. • , 0 ~ c ~
0 •• to JI o (0 0 of E' ~. o 0 0 •
00 .0 ). ... Q.. o • .e: . ~
""'" ,~ '. c a
,t4 ~ .r .. :~
• <:1 . ~
•
~ '~ 0 '0 , . . 0 c::::-
• I
• 1 • I C'. q C : . #0 0
0' ~ .. Q 00 ex:) 0
: ~ roo ~. 0 .. ~~~ 0 . ~ • • 0 ,0' 0 . ,
j~t, .~
t·
B.O 6.0 4.0 2.0 D1 (ppm)
Fig. 2.8 : NOESY (300 ms) spectrum of NKA-Iow DPe.
86
9.0
I II I II
I T31 I II
P6 1104 I II I. 11'/
I II
I~ I II
I :0 I II
II
~ I II I II
I II
I II
I II
I II
II
II
I~ W
I II
I II
~) II ~III
... -/ I II
I Itr I d I II
I II
U :: I II
I II
I II
:~ ~I;~ O~'. ... ( "
~'
I :~)
I I I I
Gal I I
IS5
01
I I
I I
I I I I
I I
I I
I I
~: I I
I I
I I
I
I
I ~50 tl I
I I I I
I Ij I
I I I
I I
I II IL9 1,. II
vf II M10
I II I
~:~ I II
I II
II
II I II
I~ I~
11)' Ic., I -
I I
I II
I II
:1 I II
II
II
I II I II
~1" ijll I II I II
II
II I II
I II
~~ I i'lJ I II
lei :"f d I~ I II
J II
B.1
D1 (ppm)
II
<0
o
I'
I ~
7.2
Fig 2.9 Fingerprint region of TOCSY & NOESY of NKA-Iow DPC TOCSY cross-peaks are shown in black and NOESY peaks in cyan. Spin systems are shown with a dotted line.
87
L9JM10-N~ C'\I
0 0 ......:
F6NH-F6HD* L9JM10-NH3
~ .,
i co ......:
~ ,B,
IJ I '"
[)~ ~
cti
10 ~
0 I fJlo/ t1 ~
c .. I 0) U
V ~
, \ ~
9.0 8.4 7.8 7.2 D1 (ppm)
Fig. 2.10: Overlay ofNH-NH regions of TOCSY (black) and NOESY (cyan) spectra of NKA-Iow DPC. NOESY cross-peaks are labelled to show sequential connectivities. The unassigned TOCSY peaks are contaminants from DPC (also reported in Kallick, et.al, 1995)
88
E' 0.. ~ C\I a
Fig. 2.11: Resonance Assignments ofNKA in d-DPC (1:4). Chemical shifts are relative 10 Tetramethylsilane, All values are in parts per million (ppm)
residue NH a p others
mSn 4.366 3.410 7.463, 8.695 LYS 9.070 4.455 t852 1.703 1.496 2.995 7.665 THR 8.419 4.297 4.263 1.239 ASP 8.436 4.636 2.759 SER 8.169 4.434 3.860,
3.779 PHE 8.560 4.502 3.154 7.260 (0$) VAL 8.007 3.864 2.102 0.966(1*) GLY 8.260 3.923 LEU 7.924 4.245 1.789 1.614 (y), 0.972(01), 0.918(01) MET 7.931 4.372 2.055 2.459 (yl), 2.580 (y2), 2.122 (E)
89
<> F6
o V7 8.9
o G8
8.8
8.7
8.6
~ f l\
8.5
8.4
8.3
~ 8.2 l ~ 8.1
8.0
280 290 300 310 320
temperature (K)
I
8.0
ppm
Fig. 2.12: The calculation of temperature coefficients of NH protons in NKA-Iow DPe: (left) Amide region of I-D spectrum ofNKA-low DPC, showing the
change in chemical shift with temperature (left top to bottom - 313, 308, 303, 298, 293, and 288 K). Panel on right shows the plot of amide chemical shift as a function of time. The slope of each function would correspond to the amide proton's temperature coefficient.
90
Fig. 2.13: Temperature coefficients of NKA-Iow DPC. The values are calculated at each step of an increase in 5K from 288.
temp- 288-293 298-298 298-303 303-308 308-313 residue HI (no NH peak) L2 4.6 5.8 7.0 T3 5.2 7.8 5.2 4.6 (2.6) D4 2.0 6.6 3.4 5.2 S5 1.6 4.6 4.0 3.2 3.0 F6 5.4 7.0 7.0 3.2 7.0 V7 4.8 5.4 6.6 4.2 4.4 G8 5.0 4.2 6.2 4.0 4.6 L9IMI0 (Have overlapping peaks at 298K)
Fig. 2.14 NH-a Coupling constants ofNKA-low DPC. . The values shown are extracted from the DQF-COSY using the program FELIX (MSI, San Diego)
Amino acid 3JNH-Cha 3JCHa-CHb
His Lys 10.606 16.363 Thr 8.869 14.094 Asp 9.032 Ser 8.867 14.874 Phe 10.020 16.571 Val 8.915 14.774 Gly -11.48 16.330 Leu 9.259 16.935 Met 9.556 17.332
91
'A 0 o~
'1 UI O.
tI ~" . ..
00 0
0 • :. ;I 00 0
o I • ~j :/
'" i ,. -./ .. - .0 H
\
~ ~·t~ ~
7 I~; ..
/ .
J> D
0 0
0 oG
j)
B.O 6.0 4.0 2.0 D1 (ppm)
Fig. 2.15 TOCSY spectrum ofNKA (2 mg) in DPC (60 mg) (1:30 ratio] (henceforth referred to as NKA -high DPC). The residual solvent is removed by deconvolution during processing
0 C'..i
o· -~ E
Q. .s: C\.I
0 a
-<0
0 o;:i
92
.? ,
8.0 6.0 4.0 2.0
D1 (ppm)
Fig. 2.16: NOESY spectrum of NKA-high DPC in D20. (As the concentration ofNKA is low, this sample was prepared by lyophilising the water and replacing it with D20 to extract more infonnation about side-chain interactions.)
93
I j II ~I I q I
III D~ \(7 I
f~ II I I
I I IIGB I UJIM10 CO 17f3 1\1 I I I c:) I I II Sp I I.
I I I~ I ~ W 8 I I 11 Y I
I II
hll I II I I
I I II I I
I I II I I L9 (0
I I II I Q> ~
I I II I Y I I II : JIl ~ ~
I f II I I
I I II I I ~
Ie ~(~ " ~ II
I I II I I M10 ~ I I II I I C\i E' I I II I ~1 y2 I I II Q.. I I II ~ yl -S:
o I I~ 0 I I C\I I I, I a I I II I
I I II I I
~,: II I I
~ C\I
II ~ I M II
I I II I I
~ I II I
I II I
I I II I I I I II I I
~ I i t~j1 ~I 0 I I , 0 I 0 l~ I ':
~ I I 'i a I I I'
I I II I I L9
~t~~ : a
o I II ~h I a
q' ~ II M10 a I d)1
I I II I I
9.0 8.1 7.2
D1 (ppm)
Fig. 2.17 Overlay of the fingerprint regions of TOCSY (black) and NOESY (cyan) (300 ms) for NKA-high DPC. Vertical lines join peaks from a spin system,
94
J
of!
@
9.0
,I I
'" L9-CONHQ ./ ,, -, '
H1HO-H1HE
8.4
/a
7.8
01 (ppm)
cO
a
C'\I
'"
Q:)
'" {,
~
cO il
7.2
Fig. 2.18: Overlay ofNH-NH regions of TOCSY (black) and NOESY (cyan) spectra of NKA-high DPC. NOESY cross-peaks are labelled to show sequential connectivities. The unassigned TOCSY peaks are contaminants from DPC (also reported in Kallick, et.al, 1995)
95
E' Q. -3: ~ a
Fig 2.19: Resonance Assignments ofNKA in d-DPC (1:30). Chemical shifts are relalive to Telramelhylsilane, All values are in parts per million (ppm)
NH a b others Residue HISn 4.413 3.456 8.656, 7.497 LYS 4.489 1.891 3.049, 1.763, 1.523 THR 8.460', 4.318 4.318 1.267 ASP 8.344 4.621 2.769 SER 8.174 4.456 3.795,
3.853 PHE 8.529 4.536 3.282, 7.286(8*), 7. 143(E)
3.171 VAL 8.011 3.885 2.153 1.059(y 1), 1.009(y2) GLY 8.320 3.962 LEU 7.904 4.269 1.858 1.649(y), 1.005(81), 0.957(81) MET 7.899 4.401 2.092 2.6139(y1), 2.489(y2), 2.187(E)
96
, 0
I
9.0
I I I G10 11 II I ,
Vi? I I I ' IIIF8 L111 I
I I S5 I ~7 ' II I K4 E
I
I .) I I II II J9 I "',4 I q6 II M1211 I
I
I
I I I I II II I I
~R I I I I I II @ I
!JJI1 I I I I I II I I
I I I I I I
I I I I I I
I I I I I I
I I I I I I
I GJ I I II I I I I II
I 0'0 I I II
: (~ : : :: : : : : ~:
@ I I I I II
I I I I cell I I I I III
I I I I III
I I I I II/I
I I I I III
I I I I III
I I I I I II
II ~ I II
I ID j'-~~ III
I I I I I II
I I I ~~
: :.: : ~ I I I
I I I
I I I
I I I
III
III
III
III
I I I III
I I I III
: :, ~: I I I 111 II I ~I
: ~~ ~: -A ," III "IV I I Il-!,
I I I 11'1 I I I III , I II
I
II I I
II I I
II I I
II I I
II I (rnJ 'Ui I 'W If I c:'~
~: lJ II I I
W:~ II I
II I
.@) I
,~ : II I
II I
II I
"O~n II I~ II I I
II I I
II I I
I I I
II I II I
II I
II I
II~ II'~
II I
II I ,: o~ I
II I
II I
II I
8.1
01 (ppm)
, 10
I
7.2
co - 0
Fig. 2.20: Overlay of fingerprint regions of TOCSY (black) and NOESY (yellow) 300 ms of Kassinin in DPC solution. Vertical lines join peaks from a spin systems
97
Fig 2.20a: Resonance Assignments of Kassinin in d-DPC. Chemical shifts are relative to Telramelhylsilane. All values are in parIs per million (ppm)
residue NH a. ~ others ASP 4.381 2.897,
2.769 VAL 8.681 4.371 2.128 1.053, 0.998 PRO 4.470 2.369, 2.034y, 3.91781, 3.69182
1.911 LYS 8.572 4.282 1.861 1.536y, 1.7388, 3.045E, 7.665NH SER 8.459 4.297 3.912,
3.878 ASP 8.350 4.670 2.880 GLY 8.242 4.208 1.960 2.221 PHE 8.202 4.480 3.212, 7.256
3.099 VAL 7.867 3.927 2.132 0.998 GLY 8.247 3.922 LEU 7.951 4.267 1.620 1.792y, 0.9748 MET 7.975 4.396 2.073 2.483yl, 2.596y2, 2.147E
98
daN(i,i+4)
da /3(i,i+ 3)
daN(i,i+3)
dNN(i,i+2)
daN(i,i+2)
dNN
2
1
0
-1
-2
H KTDS FVGlH ---T--r-----r--------r--l-I----l
I i
HKTD S FVGLM
r--r-l
00 0 0 0 _..1
\ I , ~ ! ,
~ I~ __ I ___ J
H K T 0 S F V G L M
Fig. 2.21 Secondary structure from NMR parameters. (Top) Using the NOE patterns which are unique for secondary structure. (Wuthrich, 1986). The is the probability ofa ~ tum from T3-V7. (Bottom) Using the chemical shift index method of Wishart et al (1992). Shows no indication of secondary structure.
99
Intraresidue 7 VAL
1 HIS HN 7 VAL HA 3.20
HA 1 HIS HD2 5.00 HN 7 VAL HB 2.90
HD2 1 HIS HE1 5.00 HN 8 GLY HN 3.50
2 LYS HA 7 VAL HB 3.10
HN 2 LYS QG 6.00 HA 8 GLY !iN 2.90
HA 2 LYS QG 5.60 HB 8 GLY HN 3.10
HA 2 LYS QD 6.00 8 GLY
QG 2 LYS QD 6.40 HN 8 GLY QA 3.50
QD 2 LYS QE 7.00 HN 9 LEU HN 3.30
3THR 9 LEU
HN 3 THR QG2 3.40 HN 9 LEU HA 3.20
7 VAL HN 9 LEU HB3 3.20
HA 7 VAL QQG 4.30 HA 9 LEU HB3 3.10
HB 7 VAL QQG 4.80 HA 10 MET QB 5.00
9 LEU 10 MET
HN 9 LEU HG 3.10 HN 10 MET HA 3.50
HN 9 LEU QD1 3.40 HN 10 MET QB 4.20
HN 9 LEU QD2 4.60 3 THR
HA 9 LEU HG 3.50 HN 6 PHE HN 3.30
iiA 9 LEU QD2 3.40 4 ASP
HB3 9 LEU HG 2.60 HN 6 PHE HN 5.00
HB3 9 LEU QD2 3.40 HN 8 GLY HN .3.70
HG 9 LEU QD2 3.40 5 SER
10 MET HN 7 VAL HN 3.80
. HN 10 MET HG2 4.10 HA 7 VAL HN 3.90
HN 10 MET HG3 4.20 6 PHE
HN 10 MET QE 4.00 HA 9 LEU HN 3.80
HA 10 MET HG2 4.20 HA 10 MET HN 3.80
HA 10 MET QE 3.80 7 VAL
RG3 10 MET QE 4.30 HA 10 MET HN 3.30
sequential middle and long range 1 HIS
1 RIS
HA 2 LYS HN 4.70 HD2 2 LYS QE 6.00
2 LYS HD2 5 SER HB2 5.00
UN 2 LYS HA 5.00 HD2 7 VAL HA 5.00
UN 2 LYS QB 5.50 HD2 10 MET QB 6.00
HA 3 THR HN 3.20 HE1 3 TRR QG2 4.40
3THR HE1 5 SER UN 5.00
HN 4 ASP HA 4.10 HE1 7 VAL HN 5.00
4 ASP HE1 7 VAL QQG 7.40
UN 4 ASP HA 4.50 HE1 9 LEU QD1 3.70
UN 4 ASP QS 4.20 2 LYS
UN 5 SER UN 3.40 QE 10 MET QB 7.00
HA 4 ASP QS "1.60 3 THR
HA 5 SER UN 4.10 UN 7 VAL QQG 7.40
QB 5 SER UN 5.00 UN 9 LEU QD1 3.70
5 SER QG2 5 SER HB3 5.50
UN 5 SER HA 3.60 QG2 6 PHE f9I 3.90
UN 5 SER HB2 3.40 QG2 6 PHE HA 5.40
UN 5SER HB3 3.70 QG2 7 VAL QQG 6.10
UN 6 PHE UN 3.60 QG2 9 LEU HN 4.10
HA 5 SER H83 3.50 QG2 9 LEU QD1 7.00
HA 6 PRE UN 3.20 QG2 10 MET HN 5.70
HB3 6 PHE HN 3.90 QG2 10 MET HA 5.50
6 PHE 4 ASP
UN 6 l'HE HA 3.40 QB 10 MET QB 7.00
UN 7 VAL HN 3.50 6 PHE
HA 6 PRE QB 4.70 HA 7 VAL QQG 7.40
HA 7 VAL HN 3.70 8 GLY
QS 7 VAL HN 4.90 HN .9 LEU HG 4.30
Fig. 2.22: Conformation determination of NKA-Iow DPC using DIANA. . Fig.2.22.A - Restraints generated by CALmA, a supporting program in the
DIANA package.
100
Overview: Number of accepted structures 50 (50 structures started)
Residue range for upper limits 10 lower limits 10 van der Waals: 10
Cutoff for upper limits 0.10 A lower limits 0.10 A van der Waals 0.10 A angle constraints 1.00 deg
CPU time 6.01 min CPU time per structure 0.12 min Average number of iterations 1199
struct target upper limits lower limits van der waals torsion angles
function /I sum max /I sum max • sum max II sum max
1 26 0.40 5 1.5 0.33 0 0.0 0.00 1 0.8 0.16 0 0.0 0.0
2 30 0.44 6 1.8 0.38 0 0.0 0.00 1 0.4 0.10 0 0.0 0.0
3 41 0.47 6 1.6 0.42 0 0.0 0.00 1 0.7 0.16 0 0.0 0.0
4 50 0.51 7 1.8 0.33 0 0.0 0.00 1 1.2 0.13 0 0.0 0.0
5 5 0.52 5 1. 7 0.43 0 0.0 0.00 1 0.6 0.14 0 0.0 0.0
6 17 0.56 6 1.9 0.42 0 0.0 0.00 3 0.9 0.14 0 0.0 0.0
7 21 0.56 5 1.4 0.42 0 0.0 0.00 5 1.1 0.15 0 0.0 0.0
8 31 0.57 8 2.0 0.42 0 0.0 0.00 1 0.8 0.13 0 0.0 0.0
9 9 0.57 3 1.3 0.42 0 0.0 0.00 4 1.3 0.20 0 0.0 0.0
10 8 0.57 7 1.7 0.42 0 0.0 0.00 3 1.2 0.12 0 0.0 0.0
11 20 0.61 6 1.9 0.42 0 0.0 0.00 4 1.2 0.15 0 0.0 0.0
12 43 0.63 8 2.1 0.42 0 0.0 0.00 1 0.8 0.11 0 0.0 0.0
13 44 0.64 5 1.8 0.43 0 0.0 0.00 1 1.2 0.19 0 0.0 0.0
14 48 0.65 8 2.4 0.42 0 0.0 0.00 0 0.9 0.09 0 0.0 0.0
15 38 0.72 8 2.1 0.42 0 0.0 0.00 4 1.3 0.15 0 0.0 0.0
16 4 0.73 11 2.7 0.32 0 0.0 0.00 2 1.4 0.12 0 0.0 0.0
17 18 0.73 9 2.4 0.43 0 0.0 0.00 1 0.9 0.15 0 0.0 0.0
18 28 0.76 11 2.6 0.42 0 0.0 0.00 1 0.8 0.13 0 0.0 0.0
19 15 0.80 8 1.9 0.43 0 0.0 0.00 4 1.7 0.23 0 0.0 0.0
20 29 1.09 13 3.0 0.42 0 0.0 0.00 4 2.1 0.29 0 0.0 0.0
21 40 1.14 13 3.4 0.42 0 0.0 0.00 4 1.6 0.27 0 0.0 0.0
22 24· 1.21 8 2.6 0.53 0 0.0 0.00 6 2.0 0.23 0 0.0 0.0
23 10 1.22 6 2.5 0.70 0 0.0 0.00 1 0.6 0.11 0 0.0 0.0
24 47 1.23 10 2.7 0.42 0 0.0 0.00 5 2.0 0.40 0 0.0 0.0
25 1 1.33 11 2.7 0.42 0 0.0 0.00 8 2.4 0.28 0 0.0 0.0
26 49 1.39 12 2.9 0.47 0 0.0 0.00 8 2.3 0.27 0 0.0 0.0
27 46 1.39 15 3.3 0.42 0 0.0 0.00 7 2.5 0.32 0 0.0 0.0
28 45 1.50 11 3.0 0.54 0 0.0 0.00 9 2.5 0.24 0 0.0 0.0
29 25 1. 51 10 3.5 0.49 0 0.0 0.00 3 1.9 0.17 0 0.0 0.0
30 39 1.51 12 3.4 0.56 0 0.0 0.00 7 2.4 0.21 0 0.0 0.0
31 42 1.56 10 3.5 0.52 0 0.0 0.00 2 1.7 0.12 0 0.0 0.0
32 16 1.57 8 3.0 0.70 0 0.0 0.00 3 0.8 0.25 0 0.0 0.0
33 6 1.60 12 3.7 0.70 0 0.0 0.00 2 1.1 0.20 0 0.0 0.0
34 2 1.69 16 4.0 0.46 0 0.0 0.00 6 2.2 0.26 0 0.0 0.0
35 34 1.73 11 3.2 0.58 0 0.0 0.00 6 2.3 0.49 0 0.0 0.0
36 11 1. 76 10 3.6·0.71 0 0.0 0.00 4 1.9 0.21 0 0.0 0.0
37 14 1.78 15 4.2 0.42 0 0.0 0.00 5 2.2 0.27 0 0.0 0.0
38 3 1.87 11 3.4 0.44 0 0.0 0.00 11 2.9 0.29 0 0.0 0.0
39 37 1.87 9 3.0 0.57 0 0.0 0.00 10 3.0 0.26 0 0.0 0.0 40 12 1.89 12 3.4 0.42 0 0.0 0.00 9 3.3 0.28 0 0.0 0.0 41 36 2.57 12 3.9 0.51 0 0.0 0.00 20 4.6 0.33 0 0.0 0.0 42 19 2.86 12 4.8 0.68 0 0.0 0.00 7 2.7 0.33 0 0.0 0.0 43 27 2.91 15 5.0 0.70 0 0.0 0.00 7 2.6 0.39 0 0.0 0 .. 0 44 35 3.32 13 4.4 0.70 0 0.0 0.00 20 5.0 0.40 0 0.0 0.0
45 7 3.57 13 4.0 0.52 0 0.0 0.00 26 6.0 0.36 0 0.0 0.0
46 32 4.07 12 4.0 0.57 0 0.0 0.00 22 6.7 0.61 0 0.0 0.0 47 22 4.79 16 6.3 0.66 0 0.0 0.00 22 5.5 0.32 0 0.0 0.0
48 13 4.79 18 6.2 0.67 0 0.0 0.00 19 6.1 0.43 0 0.0 0.0
49 23 5.00 14 5.8 0.93 0 0.0 0.00 20 4.9 0.36 0 0.0 0.0 50 33 9.75 23 9.3 0.81 0 0.0 0.00 42 10.4 0.54 0 0.0 0.0
Average 1. 74 10 3.2 0.51 0 0.0 0.00 7 2.3 0.24 0 0.0 0.0 +/- 1.64 4 1.5 0.13 ·0 0.0 0.00 8 1.9 0.12 0 0.0 0.0
Minimum 0.40 3 1.3 0.32 0 0.0 0.00 0 0.4 0.09 0 0.0 0.0
Maximum 9.75 23 9.3 0.93 0 0.0 0.00 42 10.4 0.61 0 0.0 0.0
Fig. 2.22: DIANA summary output: (col. I, serial number; col. 2, confonnation numbereed according to iteration, coD. target function; other columns contain details of the run )
101
Constraint violation and hydrogen bond overview (structures ordered):
Cutoff for target function Number of structures included Number of violated constraints Number of consistent violations: Maximal hydrogen bond length Maximal hydrogen bond angle Number of hydrogen bonds Number of consistent H-bonds
Upper HB3 Upper HB3 Upper HD2 Upper HEI Upper HEI Upper HEI Upper HEI Upper HA Upper QE Upper lIN Upper liN Upper HA Upper HA Upper HB Upper QG2 Upper QG2 Upper QG2 Upper QG2 Upper QG2 Upper liN Upper QS Upper liN Upper liN Upper liN Upper HA Upper HA Upper HB3 Upper HA Upper liN Upper liN Upper HB Upper liN Upper liN Upper liN Upper liN Upper lIN Upper HA Upper HA Upper HB3 Upper HB3 Upper liN Upper liN Upper liN
HIS HIS HIS HIS HIS HIS HIS LYS LYS THR THR THR THR THR THR THR THR THR THR ASP ASP SER SER SER SER SER SER PHE VAL VAL VAL GLY GLY LEU LEU LEU LEU LEU LEU LEU MET MET MET
- liN THR - QB ASP
1 - QE LYS 1 - QG2 THR 1 - liN SER 1 - liN VAL 1 - QQG VAL 2 - lIN THR 2 - HB2 MET 3 - QG2 THR 3 - HA ASP 3 - liN SER 3 - HB2 MET 3 - QB ASP 3 - HB3 SER 3 - lIN PIlE 3 - HA PRE 3 - liN MET 3 - HA MET 4 - liN PHE 4 - HB2 MET 5 - HB2 SER 5 - HB3 SER 5 - lIN VAL 5 - liN PHE 5-HN VAL 5 - liN PRE 6 - liN MET 7-HB VAL
- lIN GLY 7 - liN GLY 8-HG LEU 8 - liN MET 9 - HE3 LEU 9 - HG LEU 9 - QDI LEU 9 - HG LEU 9 - QD2 LEU 9 - HG LEU 9 - QD2 LEU
10 - HB2 MET 10 - HB3 MET 10 - HG2 MET
3 4 2 3 5 7 7 3
10 3 4 5
10 4 5 6 6
10 10
6 10
5 5 7 6 7 6
10 7 8 8 9
10 9 9 9 9 9 9 9
10 10 10
1.00E+0 50
248 o
2.40 A 35.00 deg
35 o
5 10 15 20 25 30 3~ 40 +.f .. t -f'"
max 0.53 0.67 0.30 0.67 0.42 0.64 0.43 0.30 0.39 0.81 0.57 0.73 0.59 0.30 0.23 0.66 0.58 0.27 0.57 0.27 0.57 0.23 0.21 0.42 0.11 0.41 0.39 0.70 0.76 0.66 0.26 0.58 0.54 0.58 0.93 0.29 0.71 0.47 0.43 0.38 0.38 0.28 0.60 +
+ -f -f -f~.+ ++++++~ +*++
+
+ t t
tttt
t
+ t +
+ .. + +t
++
+ +
++ +-+ ++ + +tt+
tt +
++
t
++ +
... +t +'" + +t + +t .. tttt t+t
o + + t+
... ttt++ + ... + ft+
+ ... + ... ttt + ++ ..
.+
+ • +
++
H ++
++ +
+ + • + "
J. *. "I'
it-f++ .. +t ++ <to-
·t +tt it .0 +
."+"++i"*+ +t ++t*+++
++ .. + +
t ..... * ... + +0 +
++ 0+
++ .. + +t.,.+t+
+ 0
+i + ....
itt ..
t
+++
t + f .. tttt'" ++ +tt .. • ttt .....
+ f
+ + t ttt +t
... ttt +tt ++ ttt ttttt+
tttttt++++++"'+++++++++ tttttt++
++
++ +
++ + it+t'" + it ......
+t tttttttt++ + ...
+ ++ f 0
t tt +
+
+ t
++
f
++ -++++
t +t
.. ..... + +tott... ... ..
Fig. 2.22C - Restraint violations using DIANA. The first two columns list the assignments of the cross-peak., a + against the column numbered 1-50 marks that the restraint is violated more than the cutoff . Consistant violations are seen as a string (+). In the above run, the major violations are due to inconsistant NOE intensities due to overlaps.
102
PHI and PSI Angles of five best structures after FELIX analysis
Structure 026 1 HIS PHI 2 LYS PHI 3 THR PHI 4 ASP PHI 5 SER PHI 6 PRE PHI 7 VAL PHI 8 GLY PHI 9 LEU PHI
10 MET PHI • Structure 030
1 HIS PHI 2 LYS PHI 3 THR PHI 4 ASP PHI 5 SER PHI 6 PHE PHI 7 VAL PHI 8 GLY PHI 9 LEU PHI
10 MET PHI • Structure 041
1 HIS PHI 2 LYS PHI 3 THR PHI 4 ASP PHI 5 SER PHI 6 PRE PHI 7 VAL 'PHI 8 GLY PHI 9 LEU PHI
10 MET PHI • structure 050
1 HIS PHI 2 LYS PHI 3 THR PHI 4 ASP PHI 5 SER PHI 6 PRE PHI 7 VAL PHI 8 GLY PHI 9 LEU PHI
10 MET PHI • Structure 005
1 HIS PHI. 2 LYS PHI 3 THR PHI 4 ASP PHI 5 SER PHI 6 PRE PHI 7 VAL PHI 8 GLY PHI 9 LEU PHI
10 MET PHI
from DIANA, f c 4.043E-01 -67.740 PSI 168.027
-130.394 PSI 34.920 -171.799 PSI -37.706
95.102 PSI -42.743 -115.788 PSI -34.772
57.522 PSI 13.463 -130.785 PSI -95.629 -75.135 PSI -69.428 -46.783 PSI -32.390 -63.958 PSI -107.972
from DIANA, f c 4.385E-01 -41.327 PSI 150.559
-132.249 PSI 9.014 -115.662 PSI -13.681
89.723 PSI -48.597 162.196 PSI 79.949
38.398 PSI .28.497 -53.682 PSI -97.440
-132.754 PSI -42.113 -62.598 PSI -25.517 -55.570 PSI 167.678
from DIANA, f c 4.724E-01 27.243 PSI . 163.202
-166.689 PSI 43.776 -110.810 PSI -39.010
87.340 PSI 8.541 -146.733 PSI 15.158
-11'.130 PSI -11.263 -69.658 PSI -22.198
-146.663 PSI· 18.506 36.473 PSI' 69.691
-36.902 PSI -89.161 from DIANA, fc5.096E-01 -153.162 PSI -50.861
31.919 PSI 43.108 -112.435 PSI -35.591
84.983 PSI 9.880 -150.946 PSI 15.014
-59.783 PSI -12.598 -51.223 PSI -38.275
-164.165 PSI -40.622 -58.396 PSI -15.517 -91.031 PSI -146.091
from DIANA, t-5.160E-01 -114.830 PSI -164.474 -127.174 PSI 19.738
175.360 PSI -33.750 83.952 PSI 8.340
-153.283 PSI 18.407 -62.061 . PSI -10.997 -39.070 PSI ~40.530
-158.169 PSI -98.877 -91. 754 P.SI 61. 040 -40.571 PSI -88.966
Fig. 2.22D. Dihedral angles of best 5 structures generated by DIANA
103
Fig. 2.23 Restraints for NKA-Iow DPC generated by FELIX (coil and 2 are the crosspeak assigllinents, 3 & 4, the lower and upper restraint and the last column is the class of restraint. Qualitative restraints are for those peaks that contain multiple assignments or overlapping peaks.)
(The list is continued on the next page)
1:PHE_6:HD* 1:GLY_8:HN 1:SER....5:HN 1:VAL_7:HN 1:PHE_6:HA 1:SER....5:HA 1:SER....5:HB2 1:PHE_6:HB* 1 :HISn_l :HB* 1:LYS_2:HB-1:SER....5:HN 1 :ASP _ 4 :HB* 1:VAL_7:HN 1:LEU_9:HN 1 :GLY~8 :HA* 1:VAL_7:HA 1:VAL_7:HB 1:VAL_7:HN 1:ASP_4:HA 1:SER....5:HA 1:SER....5:HBl 1:SER....5:HB2 1:ASP_4:HB* 1:PHE_6:HA 1:SER....5:HA 1:VAL_7:HA 1 :PHE_6 :HB* 1:VAL_7:HB 1:PHE_6:HD* 1 :GLY_8 :HA* 1:VAL_7:HA 1 :MET_I0 : HE-1:LEU_9:HG 1:VAL_7:HG* 1:PHE_6:HA 1:PHE_6:HB* 1:ASP_4:HN 1:ASP_4:HB-
" 1:PHE_6:HB* 1:LYS_2:HB-1:LYS_2:HG-1:SER_5:HB1 1 :SER....5 :HB2 1 :HISn_l :HB* l:MET_IO:HGl 1 :MET_IO :HE-1 :MET_IO :HB* 1 :1l'fR-3 :HG2* 1:LEU_9:HN 1:LEU_9:HG 1 :LEU_9 :HD2-1 :LEU_9 :HDl* 1 :LEU_9 :HD2-1 :SER....5 :HB2 1:VAL_7:HB 1:LEU_9:HD1* 1 :VAL_7 :HG* 1:SER_5:HA 1:LYS_2:HD* 1:LYS_2:HG* l:MET_IO:HGl 1:MET_10 :HE* 1 :MET:...IO:HB*
1:PHE_6:HN 1:PHE_6:HN 1:PHE_6:HN 1:PHE_6:HN 1:PHE_6:HN 1:PHE_6:HN 1:PHE_6:HN 1:PHE_6:HN 1 :1l'fR-3:HN 1:~3:HN 1 :ASP_4:HN 1 :ASP_4:HN 1:GLY_8:HN 1:GLY_8:HN 1:GLY_8:HN 1:GLY_8:HN 1:GLY_8:HN 1 :SER....5:HN 1:SER....5:HN 1:SER....5:HN 1:SER....5:HN i.:SER....5:HN 1:SER....5:HN 1:VAL_7:HN 1:VAL_7:HN 1:VAL_7:HN 1:VAL_7:HN 1:VAL_7:HN 1:LEU_9:HN 1:LEU_9:HN 1:LEU_9:HN 1:LEU_9:HN
"1:LEU_9:HN 1:PHE_6:HD-1:PHE_6:HD-1:PHE_6:HD* 1:ASP_4:HA 1:ASP_4:HA 1:PHE_6:HA 1:LYS_2:HA 1:LYS_2:HA
" 1:SER....5:HA 1:SER....5:HA 1 :HISn_l:HA 1 : ME'C 10 :HA 1 : MET_I 0 :HA l:ME'CIO:HA 1:lHR_3:HB 1:LEU_9:HA 1:LEU_9:HA 1:LEU_9:HA 1:LEU_9:HA 1:VAL_7:HA 1 :SER_5:HBl 1:VAL_7:HA 1:VAL_7:HA 1 :VAL_7:HA 1 : PHE_6 :HB* 1 :LYS_2:HE* 1:LYS_2:HE* 1 : MET_I 0 :HG2 1 :MET_IO :HG2 1 : ME'CI 0 :HG2
-1.0 6.0 2.5 3.5
-1.0 6.0 1.0 2.5
-1.0 6.0 -1.0 6.0 2.5 3.5 1.0 2.5 3.5 5.0 2.5 3.5
-1.0 6.0 -1.0 6.0 -1.0 6.0
1.0 2.5 -1.0 6.0 -1.0 6.0 2.5 3.5
-1.0 6.0 2.5 3.5 1.0 2.5
-1.0 6.0 -1.0 6.0 2.5 3.5
-1.0 6.0 -1.0 6.0 -1.0 6.0 2.5 3.5
-1.0 6.0 2.5 3.5
-1.0 6.0 -1.0 6.0 -1.0 6.0
1.0 2.5 3.5 5.0 2.5 3.5 2.5 3.5 2.5 3.5 2.5 3.5
-1.0 6.0 -1.0 6.0 3.5 5.0
-1.0 6.0 -1.0 6.0
2.5 3.5 2.5 3.5
-1.0 6.0 -1.0 6.0 1.0 2.5 1.0 2.5 1.0 2.5
-1.0 6.0 -1.0 6.0 -1.0 6.0 -1.0 6.0
1.0 2.5 -1.0 6.0 -1.0 6.0 3.5 5.0 2.5 3.5 3.5 5.0 1.0 2.5
-1.0 6.0 -1.0 6.0
qualitative medium qualitative strong qualitative qualitative medium strong weak medium qualitative qualitative qualitative strong qualitative quali tati ve medium qualitative medium strong qualitative quali tative medium qualitative quali tative quali tative medium qualitative medium qualitative que.1itative qualitative strong weak medium medium medium medium quali tative qualitative weak " qualitative qualitative medium medium qualitative qualitative strong strong strong qualitative quali tative quali tative qualitative strong qualitative qualitative weak medium weak strong quali tati ve qualitative
104
1:MET_I0:HE- 1 : MET":' 10 :HG 1 -1.0 6.0 qualitative 1 :MET_IO :HB- 1 :MET_IO :HGI -1.0 6.0 quali tati ve 1 :MET_I0 :HB- 1 :MET_I0 :HE- -1.0 6.0 qualitative 1:VAL_7:HGe 1:VAL_7:HB -1.0 6.0 qualitative 1:LEU_9:HN 1 :MET_I0 :HB- 2.5 3.5 medium 1: LYS_2 :HGe 1:LYS_2:HB- -1.0 6.0 qualitative 1:LEU_9:HDl- 1:LEU_9:HB2 :-1.0 6.0 qualitative 1 :LEU_9:HD2- l:LEU 9:HB2 -1.0 6.0 qualitative 1:LYS_2:HB- 1: L YS=:2 :HD- -1.0 6.0 qualitative l:LYS_ 2:HGe 1:LYS_2:HD- -1.0 6.0 quali tative 1: LEU_9 :HD2- 1:LEU_9:HG -1.0 6.0 qualitative l:VAL_ 7:HN 1 :VAL_7 :HGe 2.5 3.5 medium 1:LEU_9:HN 1: LEU_9 :HD2- 2.5 3.5 medium 1:GLY_8:HN 1:PHE_6:HB- 3.5 5.0 "eak 1:LEU_9:HN 1:PHE_6:HB- -1.0 6.0 qualitative 1 :SEJt..5:HN 1:PHE_6:HB- 3.5 5.0 weak 1:LYS_2:HN 1 :HISn_l :HB- 3.5 5.0 "eak 1 :HISn_l :HD2 1 :HISn_l :HB- 2.5 3.5 medium 1:LyS_2:HN 1 :HISn_l:HA 3.5 5.0 "eak 1:PHE_6:HN 1:ASP,,:,4:HA 3.5 5.0 weak 1:GLY_8:HN 1:LEU_9:HBl 3.5 5.0 weak 1:LYS_2:HN 1: LYS_2 :HB- 3.5 5.0 weak 1:LYS_2:HN 1: LYS_2 :HGe 3.5 5.0 weak 1:VAL_7:HB 1:PHE_6:HN 3.5 5.0 weak 1:ASP_4:HB- 1:PHE_6:HN -1.0 6.0 quali tative 1:SEJt..5:HA 1 :ASP_ 4 :HB- 3.5 5.0 weak 1 :MET_IO:HA 1 :MET_IO :HG2 -1.0 6.0 qualitative 1:LYS_2:HA 1:LYS_2:HD- -1.0 6.0 qualitative 1:PHE_6:HA 1:LEU_9:HG -1.0 6.0 qualitative 1 :MET_I0:HA 1:LEU_9:HG 3.5 5.0 weak
105
Fig. 2.24 Restraints for NKA high-DPC generated by FELIX (colI and 2 are the crosspeak assignments, 3 & 4, the lower and upper restraint and the last column is the class of restraint. Qualitative restraints are for those peaks that contain multiple assignments or overlapping peaks-)
(The list is continued on the next page)
l:HISn l:HA l:HISn l:HEl -1.0 3.0 w~ak
l:THR 3:HB l:ASP 4:HA -1.0 6.0 qualitative l:LYS 2:HA l:PHE-6:HA -1.0 3.50 medium l:LEU-9:HB* -l:PHE 6:HA -1.0 3.50 medium l:PHE 6:HD* 1: PHE-6:HA -1.0 3.50 medium 1:LEU-9:HG 1 :PHE-6:HA -1.0 6.0 qualitative l:ASP -4:HA 1:SER-5:HA -1.0 3.0 weak l:PHE-6:HA 1 :SER-5:HA -1.0 3.50 medium l:SER--5:HBl 1:SER-5:HA -1.0 2.50 strong l:THR-3:HG* 1:THR-3:OO -1.0 3.50 medium 1 :VAL--7 :HGI * 1 :THR-3:HB -1.0 6.0 quali tati ve l:PHE - 6:HD* 1:LEU-9:HA 6.0 quali ta ti ve -1.0 l:MET
-. 10:HA 1:LEU-9:HA -1.0 3.50 medium
l:LEU-.9:HA l:GLY-8:HA* -1.0 3.0 weak l:PHE 6:HD* 1:SER-5:OOl -1.0 3.0 weak l:SER-5:HA 1: SEIC5:HB2 -1.0 2.50 strong l:HISn l:HA l:HISn 1:00* -1.0 3.50 medium l:LYS 2:HD* l:LYS 2:HE* -1.0 6.0 quali tati ve 1:ASP-4:HA 1:ASP-4:OOl -1.0 2.50 strong 1 :MET-10:HA 1:MET-10:HG1 -1.0 3.50 medium l:MET -lO:HE* l:MET -lO:HGl -1.0 3.50 medium - -l:MET 10:HB* l:MET lO:HGl -1.0 3.50 medium l:MET-10:HG1 l:MET lO:HG2 -1.0 2.50 strong l:MET 10:HA l:MET-lO:HG2 -1.0 3.50 medium l:MET-IO:HE* l:MET 10:HG2 -1.0 6.0 qualitative 1:MET-10:HB* 1:MET-IO:HG2 -1.0 3.50 medium 1 :MET-IO:HA l:MET-IO:HE* -1.0 3.50 medium 1:VAL-7:HA 1:ru-7:OO -1.0 3.50 mediUm l:MET-IO:HA l:MET-IO:HB* -1.0 3.50 medium
. l:LYS -2:HA l:LYS -2:HB* -1.0 3.50 medium l:LYS -2:HD* -l:LYS 2·:HB* -1.0 3.50 medium l:LYS -2:HG* 1:LYS=:2:HB* -1.0 3.50 medium 1 :LEU-9:HA l:LEU 9:00* -1.0 3.50 medium 1:LEU-9:HG 1:LEU-9:OO* -1.0 2.50 strong l:PHE -6:HB1 1:LEU-9:HB* -1.0 3.0 weak 1:LYS-2:HA l:LYS -2:HD* -1.0 3.0 weak 1:LEU-9:HA l:LEU 9:HG -1.0 3.50 medium l:LEU-9:HD2* l:LEU 9:HG -1.0 3.0 weak l:LYS 2:HE* l:LYS 2:HG* -1.0 3.0 weak -l:LYS 2:HA l:LYS 2:HG* -1.0 3.50 medium l:ASP - 4:HA l:VAL-7:HG1* -1.0 3.1) weak l:PHE 6:HD* l:LEU-9:HD1* -1.0 3.0 weak l:LEU 9:HB* l:LEU 9:HD1* -1.0 3~ ·~ium
1: LEU-9:HA l:LEU-9:HD2* -1.0 3.50 .medium l:PHE-6:HD* l:PHE 6:001 -1.0 3.50 medium 1:PHE-6:HD* 1:LEU-9:HB* -1.0 3.0 weak 1 :LEU-9:HA 1:THR-3:HG* -1.0 3.0 weak 1:VAL-7:HA 1 : VAL -7:BG2* -1.0 3.50 medium 1:VAL-7:HB 1:VAL-7:HG2* -1.0 3.50 medium 1:VAL-7:HGl* 1:VAL-7:HB -1.0 3.50 medium 1:SER-5:HA -.4:HB1 l:ASP -1.0 3.0 weak l:PHE 6:HBl l:PHE 6:HA -1.0 3.50 medium
106
l:PHE 6:HB2 l:PHE 6:HA -1.0 3.50 medium l:SER 5:HBI l:SER 5:HB2 -1.0 2.50 strong l:PHE 6:HB2 l:PHE 6:HN -1.0 6.0 qualitative -1 ::SER 5:HN l:PHE 6:HN -1.0 6.0 weak -l:VAL 7:HN l:PHE 6:HN -1.0 6.0 weak
- -6:HN -1.0 6.0 qualitative l:PHE 6:HD* 1: PHE - -
l:SER 5:HA 1: PHE 6:HN -1.0 6.0 weak -
l:SER 5:HB2 l:PHE 6:HN -1.0 6.0 qualitative -
l:PHE 6:HA l:PHE 6:HN -1.0 6.0 weak - -l:LYS 2:HA l:THR 3:HN -1.0 6.0 weak l:THR-3:HB l:THR 3:HN -1.0 6.0 weak -l:ASP 4:HN l:THR 3:HN -1.0 3.50 medium
-2:HB* --1.0 6.0 qualitative l:LYS l:THR 3:HN
l:SER-5:HN l:ASP 4:HN -1.0 6.0 weak l:THR- -
weak 3:HB l:ASP 4:HN -1.0 6.0 -
l:ASP 4:HBI 1 :ASP 4:HN -1.0 3.50 medium -l:VAL 7:HN l:GLY 8:HN -1.0 6.0 weak -l:LEU 9:HN l:GLY 8:HN -1.0 3.50 medium 1 :GLY 8:HA* l:GLY 8:HN -1.0 3.50 medium l:VAL-7:HB l:GLY - 8:HN -1.0 6.0 qualitative l:LEU-9:HDl* l:GLY - 8:HN -1.0 6.0 weak l:SER-5:HA
- 5:HN 6.0 weak l:SER -1.0 l:SER-5:HBI l:SER 5:HN -1. 0 6.0 weak l:SER-5:HB2 - 5:HN -1.0 6.0 weak l:SER l:ASP 4:HBI l:SER 5:HN -1.0 6.0 weak l:VAL 7:HA l:VAL 7:HN -1.0 3.50 medium l:VAL 7:HB l:VAL 7:HN -1.0 6.0 quali tati ve l:VAL-7:HGl* - 7:HN weak l:VAL -1.0 6.0 l:PHE 6:HZ l:LEU 9:HN -1.0 6.0 weak -l:LEU 9:HA 1 : LEU 9:HN -1.0 3.50 medium -l:GLY 8:HA* l:LEU 9:HN -1.0 6.0 weak l:LEU 9:HB* l:LEU 9:HN -1.0 6.0 qualitative l:LEU 9:HG l:LEU 9:HN -1. 0 3.50 medium l:LEU-9:HDl* l:LEU 9:HN -1.0 6.0 weak l:PHE 6:HD* l:LEU 9:HN -1.0 6.0 weak l:MET - 10:HE* l:LEU-9:HN -1.0 6.0 weak -l:PHE 6:HZ l:PHE 6:HD* -1.0 3.50 medium - -l:PHE 6:HB2 l:PHE 6:HD* -1.0 6.0 qualitative -- -l:LYS 2:HG* l:LYS 2:HD* -1.0 6.0 weak
107
11-NH
10-NH
9-NH
8-NH Q) 0 c 7-NH Q) ::J CF 6-NH Q) en
~ 5-NH
Z 4-NH
3-NH
2-NH
1-NH
~ ~®® 0 •
0 ~~ • 9 ~~®® • ®~
: ·· m~f;3 ®
0 0
• • 0 ~~o 0 ~~® ••
®9~ ~®®
®® 0 ~ • 0
• : ~f;3® NKAlow-OPC 0
~ : • NKA high-OPC
1-NH 2-NH 3-NH 4-NH 5-NH 6-NH 7-NH 8-NH 9-NH 10-NH 11-NH
NKA sequence
Fig. 2.25 Scatter-plot showing comparison of NOE peaks in NKA-low DPC (yellow circles) and NKA-high DPC (red crosses). The long range peaks are visible in the NKA high-DPC spectra, but were too noisy to measure (the peptide concentration is 2/5 that of the low-DPC sample). The extra peaks are intra-residue side chain peaks- visible in the NKA-high DPC in D20. There is no significant differences in conformation induced by a higher concentration of the lipid as there are no additional medium to long range interactions.
On the X and Y axes, each major tick marked NH is the NH proton of the amino acids in sequence (HKTDSFVGLM), the a, b and other protons of the sidechain are represented at each position of the minor tick - at 0.25 ,.0.5 and 0.75 respectively.
108
A
~,~fr;yW~~b / ~ J ,
B c D
F E
~t.
, ,_. J'
I ~ ,
... -:
Fig. 2.26: Simulated Annealing (SA) : Snapshots showing different stages of SA on NKA-high DPC. In all the above figures, the side-chains are coloured according to a hydrophobic scale in which increasing hydrophobicity is red, and polarity blue, Restraints are shown in yellow. A,: The extended conformer. B. Conformation where coordinates are randomised to remove the bias of a starting structure. C. scaling of restraints, D: scaling of covalent terms, E: during cooling. F. Final minimised structure.
109
Fig. 2.27 :Conformations of NKA-low DPC Top - Peptide backbone of 50 structures superimposed on lowest charge conformer Bottom - As above, with side chains. The side chains are coloured with increasing hydrophobicity from blue to red.
110
• '0 • • K2 • • • • •• 150 . , .. .. • T3
• • D4 .... : .. ". . 0 S5 100 . -.. . . • F6 • • • V7 • • • • 00 ..
o '" ~ '1t . r • G8
50 • :.~ y • •
0 L9 . , • M10
'¥ • 0-1&. • 0 • • • • 0 •
• • 0 0
-SO • · 0 ••
• 8· :-. .. . ~ • . at tto
-100 • o 0.~.'6 • ?a'crf6 " • -150 ..' . 0 0 );> ...
~ ~ .~ ..
-1 SO -100 -SO 0 50 100 150
l/J
Fig. 2.28 Ramachandran plot of NKA-low DPC The phi psi dihedral angles have show a large degree of flexibility for the molecules backbone.
111
Fig. 2.29 Restraints for Kassinin in DPC generated by FELIX (colI and 2 are the crosspeak assignments, 3 & 4, the lower and upper restraint and the last column is the class of restraint. Qualitative restraints are for those peaks that contain multiple assignments or overlapping peaks.)
(The list is continued on the next page)
lower upper
_1 :ASP!Ll:HA l:VAL_ 2:HN -1.00 6.00 weak .--~--
l:ASPN_ 1:HB1 1:VAL_2:HN -1.00 6.00 weak 1 :VAL_2 :HG2- l:VAL_ 2:HN -1.00 6.00 weak 1:VAL_2:HB 1:VAL_2:HN -1.00 3.500 medium l:SER_S:HN 1 :LYS_4:HN -1.00 6.00 weak 1:PRO_3:HA 1:LYS_4:HN -1.00 3.500 medium 1:ASP_6:HN 1 :SEIt-S:HN -1.00 3.500 medium 1: SEIt-S:HB 1 1 :SEIt-S:HN -1.00 6.00 weak 1:SEIt-S:HB2 l:SER_S:HN -1.00 3.500 medium l:SEIt-S:HA 1:ASP_6:HN -1.00 3.500 medium 1 :ASP _6 :HB- 1:ASP_6:HN -1.00 6.00 weak 1:GLN_7:HN 1:ASP_6:HN -1.00 3.500 medium 1 : SEIt-5 : HB 1 1:ASP_6:HN -1.00 3.500 medium 1 :LEU_ll:HN l:GLY_ 10:HN -1.00 3.500 medium 1:VAL_9:HB l:GLY_lO:HN -1.00 3.500 medium 1 :ASP_6 :HB- 1:GLN_7:HN -1.00 6.00 weak 1 :PHE_B :HB2 l:PHE_B:HN -1.00 3.500 medium 1 :VAL_9 :HG- l:PHE_ B:HN -1.00 6.00 weak 1 :MET_12 :HB- 1 :MET_J2:HN -1.00 6.00 weak 1 :MET_12 :HG2 1 :MET_12 :HN -1.00 6.00 weak l:GLY_ 10:HA- l:LEU_ll:HN -1.00 3.500 medium l:LEU_ll:HD- . 1: LEU_ll :HN -1.00 6.00 weak 1 :GLY_10:HN 1:VAL_9:HN -1.00 3.500 medium l:PHE_B:HN 1:VAL_9:HN -1.00 3.500 medium 1:VAL_9:HB 1:VAL_9:HN -1.00 3.500 medium 1 :VAL_9 :HG- 1:VAL_9:HN -1.00 6.00 weak l:PHE_ B:HB2 1:VAL_9:HN -1.00 6.00 weak 1:PHE_B:HB1 1:VAL_9:HN -1.00 6.00 weak 1:PHE_8:HA l:PHE_B:HE- -1.00 6.00 weak -I :PHE_B :HB1 l:PHE_B:HE- -1.00 6.00 weak 1:PHE_8:HB2 l:PHE_B:HE- -1.00 6.00 weak 1: PRO_3 :HD2 1:PRO_3:HA -1.00 6.00 weak 1 :PRO_3 :HD1 1:PRO_3:HA -1.00 3.500 medium 1:VAL_2:HG1- 1:PRO_3:HA -1.00 6:00 weak 1:PRO_3:HBl 1:PRO_3:HA -1.00 6.00 weak 1 :MET_12:HN 1:MET_12:HA -1.00 6.00 weak 1 :MET_12 :HB- 1 :lifE"C12:HA -1.00 6.00 weak 1:SER_S:HB1 l:SER_S:HA -1.00 3.500 medium 1:SER_S:HB2 l:SER_S:HA -1.00 3.500 medium l:LEU_ ll:HG l:LEU_ll:HA- -1.00 3.500 medium l:LEU_ 1l:HB2 1 :LEU_ll:HA -1.00 3.500 medium 1 :GLN_7 :HG- 1:GLN_7:HA -1.00 6.00 weak 1 :GLN_7 :HB- 1:GLN_7:HA -1.00 3.500 medium 1:PRO_3:HD2 1:PRO_3:HDl -1.00 3.500 medium l:PRO_ 3:HG- 1 :PRO_3 :HD2 -1.00 6.00 weak 1 :VAL_2 :HG1- 1: PRO_3 :HD2 -1.00 6.00 weak 1:PHE_8:HB2 1:PHE_8:HBl -1.00 2.500 strong 1:LYS_4:HD- 1:LYS_4 :HE- -1.00 6.00 weak l:ME"C 12:HG2 l:MET_ 12:HG1 -1.00 3.500 medium 1 :MET_12 :HE- 1 :MET_12 :HGI -1.00 6.00 weak 1: PRO_3 :HG- l:PRO_ 3:HBl -1.00 3.500 medium l:PRO 3:HB2 1:PRO_3:HBl -1.00 3.500 medium 1:GLN_7:HN 1 :GLN_7 :HG- -1.00 6.00 weak 1:GLN_7:HB- 1:GLN_7:HG- -1.00 3.500 medium l:MET_ 12:HA l:MET_ 12:HE- -1.00 6.00 weak
112
l:LEU_ 11 :HB2 l:LEU_ ll:HG -1.00 2.500 strong l:LYS_ 4:HD* 1: LYS_ 4 :HG* -1.00 6.00 weak l:LYS_ 4:HB* 1:LYS_4:HG* -1.00 6.00 weak l:ME'C 12:HN 1 :MET_12:HG1 -1.00 6.00 weak l:LYS_ 4:HN l:LYS_ 4:HB* -1.00 6.00 weak l:LYS 4:HN l:LYS_ 4:HG* -1.00 6.00 weak 1:GLN_7:HN 1:GLN_7:HA . -1. 00 3.500 medium l:MET_ 12:HN 1 : LEU_ 1l:HB2 -1.00 6.00 weak l:MET_ 12:HN l:LEU_ ll:HG -1.00 3.500 medium l:MET_ 12:HN l:LEU_ l1:HA -1.00 6.00 weak 1:PRO_3:HA l:PRO - 3:HG* -1.00 6.00 weak 1 :PRO_3:HA 1 : PRO_ 3:HB2 -1.00 3.500 medium 1:PRO_3:HD1 l:PRO_ 3:HG* -1.00 6.00 weak l:LEU_ll:HG l:LEU_ ll:HD* -1.00 6.00 weak 1:VAL_2:HN l:PRO_ 3:HA -1.00 6.00 weak l:PHE_B:HN l:PHE_B:HA -1.00 6.00 weak 1:VAL_9:HN l:PHE_B:HA -1.00 6.00 weak l:SER_S:HN 1:ASP_6:HB* -1.00 6.00 weak 1:LYS_4 :HN 1:PRO_3:HB1 -1.00 6.00 weak 1 :GLY_10:HN l:LEU_ll:HG -1.00 6.00 weak 1:SE~5:HN 1:LYS_4:HG* -1.00 6.00 weak l:SER_S:HN 1:PRO_3:HB1 -1.00 6.00 weak 1:LYS_4:HB* l:SER_S:HN -1.00 6.00 weak 1:PRO_3:HD1 1:PRO_3:HB2 -1.00 6.00 weak 1: PRO_3 :HD2 1:PRO_3:HB2 -1.00 6.00 weak 1:PRO_3:HD1 1:PRO_3:HB1 -1.00 6.00 weak 1:PRO_3:HA 1:VAL_2:HB -1.00 6.00 weak 1 :MET_12:HA 1 :MET_12 :HG2 -1.00 3.500 medium 1 :MET_12:HA l:MET_ 12:HG1 -1.00 6.00 weak 1:VAL_2:HB 1 :PRO_3 :HD2 -1.00 6.00 weak 1 :MET_12 :HG2 1 :MET_12 :HE* -1.00 6.00 weak
113
Fig. 2.30 NOE restraints of Kassinin shown mapped onto the extended conformer of the peptide. The side-chains are coloured according to a hydrophobic scale in which increasing hydrophobicity is red, and polarity blue. Restraints are shown in yellow.
113
Fig. 2.31 Conformations of Kassinin in DPC solution Top - Peptide backbone of 50 structures superimposed on lowest charge conformer Bottom - As above, with side chains the side chains are coloured with increasing hydrophobicity from blue to red.
114
• • • V2 150 • P3 • • K4
0 S5 100 • D6
• Q7
• Fa 50 0 V9 • 0 • G10
• L11 cp • M12
0
-50
• • • -100 • •
• • ~1f. e\ -150
• • It • • • -150 -100 -50 0 50 100 150
f/J
Fig. 2.32 Ramachandran plot of Kassinin in DPC solution The phi psi dihedral angles show that all conformers fall into allowed areas of the Ramachandran plot (see text for discussion)
115
;0 ro -~ ~. <:. ro -0 100 o -0 C -~ ~. o :J
-150
80 -150 -1 00 -50 o 50 100 150
Figure (2.32): The population of all trialanine conformers derived by rotating the ~ and 'V torsions of the central alanine residue. To explain the variation on the basis of flexibility for Kassinin and NKA, we have simulated all conformations of a tripeptide containing alanine residues by rotating the <I> and \jJ torsions of the central residue in steps of 10 degrees using the molecular mechanics and dynamics program }Jisc()ver R (MSI, San Diego), and mapping the energies of resultant conformations as a function of their ~,'V values .. To describe the relative populations of each conformer, a Boltzmann distribution was applied to the eneq::>"'Y map. The above figure shows the relative populations of trialanine conformers as a percentage of the lowest energy function .