Step-by-step RCI protocol
The protocol
presented here consists of several main blocks: 1) reference correction of
chemical shifts assignments; 2) calculations of sequence-specific random coil
chemical shifts; 3) calculation of the Random Coil Index; 4) prediction of model
free order parameters and root mean square fluctuations of NMR and MD
ensembles.
The performance of
the RCI method is strongly dependent on having properly referenced chemical
shift data. Users must ensure that the backbone chemical shift assignments of
the protein of interest have been correctly referenced to DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid)
according to IUPAC-IUBMB standards 17. The indirect chemical referencing method has
been described in detail in a number of publications 17,18 and the chemical referencing
process is usually indicated in most BMRB chemical shift assignment file. A
recent study revealed that 20%-30% of published assignments require reference
correction of one or more nuclei 19. If one is uncertain about the quality or
correctness of the chemical shift referencing for their protein, please include
steps 5-11 of the PROCEDURE below. These steps describe a simple procedure of
referencing correction that is based on a global minimization of the average
difference between chemical shifts in helices and b-strands of a particular protein and the chemical shifts
commonly observed in these secondary structure elements in a large protein
database.
To generate an
accurate set of RCI values it is necessary to first determine a set of
sequence-specific random coil chemical shifts (see step 2). In the current protocol, these random coil
shifts are calculated from random coil chemical shifts 20 and neighboring residue corrections 21 published by Dyson group. We
chose these reference values because they were obtained under conditions (8 M
urea, pH 2.3) that are expected to significantly diminish possible long-range
intra-peptide interactions. Also, this set of reference values is the only one
that includes i+2, and i-2 adjacent residue corrections.
Random Coil Index is
calculated as the inversed average of weighted secondary chemical shifts (see
equation 1 below). Weighting coefficients were optimized by maximizing correlation
between RCI and RMSF of MD ensembles of 14 proteins. RCI expression was then tested using a set of
four new proteins as well as leave-one-out approach and the training set of 14
proteins. Coefficients of correlation between RCI and MD RMSF were 0.82 for
both training and test sets. RCI was also found to correlate well with
model-free order parameters and RMSFs of NMR
ensembles of all 18 proteins with correlation coefficients of 0.77 and 0,81, respectively. Details of development and tests of the
RCI method have been published elsewhere 12.
Based on the good correlation between RCI and aforementioned measures of
motional amplitudes, we determined empirical expressions that can be used to
convert RCI into these parameters. These equations are presented at the end of
this protocol
Despite
the good overall agreement between RCI and protein flexibility, one should not
over-interpret RCI values at the protein termini. Since the effects of
polypeptide chain termination on random coil values and neighboring residue
corrections are not well known and not fully taken into account in RCI
calculations, the accuracy of RCI flexibility predictions is expected to be low
for these regions. While the end-effect correction step makes RCI a slightly
better predictor of protein flexibility, the improved correlation is not
related to the effects of conformational averaging on chemical shifts. Instead,
it apparently originates from the indirect correlation between the increase of
secondary chemical shifts and protein flexibility due to their shared
dependence on the proximity to the terminal residue. We generally recommend that users exclude RCI
values of the three terminal residues from consideration or, if the end-effect
correction is applied, interpret their values as the commonly observed profile
of protein flexibility at termini.
PROCEDURE
1| Obtain 13Ca, 13Cb, 13CO, 1Ha, 1HN, 15N assignments
from NMR experiments
2| Calculate the sequence-corrected random coil reference values
for the 13Ca, 13Cb, 13CO, 1Ha, 1HN, and 15N
chemical shifts by adding the neighboring residue correction factors for
residues i+1, i-1, i+2, and i-2 21 to
the random coil values of residue i 20.
3| Calculate the secondary chemical shifts for each residue in the
protein by subtracting the sequence-corrected random coil chemical shift values
from the experimental chemical shifts of the corresponding residue. For example, if the first residue is
4| If you are certain that your protein is properly referenced,
continue to step 12. If not, re-reference the set of chemical shift assignments
as described in steps 5-11.
RE-REFERENCING CHEMICAL SHIFT
ASSIGNMENTS
5| If 1Ha shifts are available, determine the protein’s secondary
structure from 1Ha Chemical Shift Index (A). If the 1Ha shifts are unavailable, replace step 5 with
step 9.
A Chemical Shift Index
(i)
Assign index values of -1,1 and 0 to residues with secondary
chemical shifts (ppm) in the respective ranges
tabulated below.
Table 1: Chemical shift indices
|
-1 |
0 |
+1 |
1Ha
(all amino acids) |
<
-0.1 |
-0.1
®
0.1 |
>
0.1 |
13Ca
(all amino acids except proline) |
<
-0.7 |
-0.7
®
0.7 |
>
0.7 |
13Ca
(proline) |
<
-4.0 |
-4.0
®
4.0 |
>
4.0 |
13CO (all except proline) |
<
-0.5 |
-0.5
®
0.5 |
>
0.5 |
13CO (proline) |
<
-4.0 |
-4.0
®
4.0 |
>
4.0 |
13Cb
(all amino acids except glycine and proline) |
<
-0.7 |
-0.7
®
0.7 |
>
0.7 |
13Cb
(proline) |
<
-4.0 |
-4.0
®
4.0 |
>
4.0 |
(ii) Referring to the table below, assign
a helical state to a group of four or more residues with ’helix’ indices not
interrupted by a residue with ’sheet’ index. Assign a b-strand state to a group of three or more residues with ’sheet’
indices not interrupted by a residue with a ‘helix’ index.
Table 2: Helix and sheet indices
|
-1 |
+1 |
1Ha
|
helix |
sheet |
13Ca
|
sheet |
helix |
13CO |
sheet |
helix |
13Cb
|
Not
applicable* |
sheet |
*13Cb secondary chemical shifts should only be
used for prediction of b-strands.
(iii) When a gap in the aforementioned
patterns occurs, use a “local density” criterion to determine secondary
structure of the gap residues. Assign either helical or b-strand state to a stretch of five residues if 70% of these
residues have ‘helix’ or ‘sheet’ indices, respectively
(iv) Identify
termination points of helices and b-strands
by the first appearance of two consecutive zero indices or indices with the
sign opposite to those of the corresponding secondary structure.
(v) If a protein region demonstrates
a pattern of chemical shift indices that is not consistent with any
aforementioned rules, the secondary structure is assigned as coil.
6| Subtract the chemical shift of each residue in identified
helices and/or b-strands from the average
chemical shift of the corresponding residue that is commonly observed in these
secondary structure elements. The values of the commonly observed chemical
shifts can be found in Supplementary Table 3 22.
7| Calculate the reference offset for each type of shift (13Ca,
13Cb, 13CO, 1Ha,
1HN, and 15N) by averaging the aforementioned differences
between experimental and commonly observed chemical shifts over all helices
and/or b-strands in the protein.
8| Add the calculated offsets to the values of the corresponding
experimental chemical shifts of each residue to correct the spectral
referencing.
9| Determine the secondary structure from the chemical shift
indices for 13Ca, 13Cb, 13CO, and 1Ha, if available (A).
10| Determine the final secondary structure assignment for the
protein using either rule A or rule B (below) depending on availability of
chemical shift assignments.
A) If three or
more types of chemical shift are available for a residue, the final prediction
is the consensus secondary structure that is predicted by the majority of shifts.
In case of b-strand prediction, if an
equal number of shifts predict b-strand
and non-b-strand structure, use the prediction
of the group of shifts without 13Cb.
Exclude non-b-strand predictions
originating from 13Cb shifts
from the calculation of the consensus, when the choice between a helical structure
and a coil structure has to be made.
B) If fewer than
three types of chemical shifts are available for a particular residue, the final
secondary structure assignment is based on the predictions by individual shifts.
Give precedence to these predictions in the following order: 13Ca, 1Ha, 13CO, and 13Cb.
11| Repeat steps 6-7. If the chemical offsets for all types of
chemical shifts are small (< 0.0001 ppm), proceed
to the next step. If not, repeat steps 8, 9 and 6-7 until the offsets become
smaller than < 0.0001 ppm.
12| Remove secondary chemical shifts of cysteine
residues.
13| Substitute 13C,
1H, and 15N secondary chemical shifts below 0.04 ppm, 0.01 ppm and 0.1 ppm, respectively, with the corresponding “floor values”
(0.04 ppm for 13Ca, 13Cb, 13CO, 0.01 ppm
for 1Ha and 1HN,
and 0.1 ppm for 15N).
14| Fill any gaps in the per-residue distributions of the secondary
chemical shifts. If a chemical shift for residue i is missing, use the average of
secondary chemical shifts of residues i+1
and i-1 as a proxy for the
secondary chemical shift of residue i. If the chemical shifts of residues i+1 or/and i-1 are
missing, include the secondary chemical shifts of residues i+2 or/and i-2 in
calculation of the average.
CRITICAL STEP This step is optional. While we consider
filling gaps in per-residue distributions of secondary chemical shifts
appropriate in the middle of a long (≥
5 residues) secondary structure element (helix, b-strand,
long loop), it may be not desirable to do so for shorter stretches of secondary
structure and at transition points between secondary structure elements with
significantly different dynamic properties (e.g. a transition from a rigid
helix to a mobile loop).
14| Apply a three-residue averaging to smooth the per-residue
distributions of the chemical shifts. This involves taking the secondary shifts
of three residues, averaging them and then assigning that average value to the
middle residue and repeating the process sequentially for each triplet of
residues in the protein. Use the average of the first two and the last two
secondary chemical shifts to obtain smoothed values of secondary shifts for the
N-terminal and C-terminal residues, respectively.
15| Multiply 13C
(13Ca, 13Cb, and 13CO)
and 1H (1Ha and 1HN)
secondary chemical shifts by 2.5 and 10.0, respectively. If the new secondary
chemical shift is below 0.5 ppm, replace it with the
“floor value” of 0.5 ppm. Also, replace 15N
secondary chemical shifts below 0.5 ppm with the
“floor value” of 0.5 ppm .
16| Determine the weighting coefficients needed to calculate the Random
Coil Index (equation 1) that correspond to the set of available secondary
chemical shifts (Supplementary Table 4). Multiply each coefficient by 7.5.
17| Calculate the Random Coil Index (RCI) using the following
expression.
RCI = (<A |DdCa | +B |DdCO |+C |DdCb |+ D|DdN | +E |DdNH | +F |DdHa |> )-1 (1)
where |DdCa |, |DdCO |, |DdCb |, |DdN |, |DdNH |, and |DdHa |
are the absolute values of the secondary chemical shifts (in ppm) of Ca, CO, Cb,
N, NH and Ha, respectively. A,B,C,D,E, and F are
weighting coefficients (Supplementary Table 4).
Left angle and right angle brackets (<
>) indicate the average. If the RCI value is above 0.6, replace its value
with the “ceiling value” of 0.6. At this point, you may wish to apply the
end-effect corrections to N- and C-termini when the RCI values of the first
or/and the last three residues progressively decrease toward the corresponding terminal residue
(B).
B End-effect corrections
(i) Identify
the largest RCI value among the four terminal residues.
(ii) Calculate the difference
between this maximal RCI value and the RCI value of each remaining terminal
residue.
(iii) Multiply the calculated
difference by two and add the result to the RCI value of corresponding residue.
If the new RCI value is above 0.6, replace it with the “ceiling value” of 0.6.
18| Apply a second three-residue averaging to smooth the per-residue
distributions of the RCI values. Use the average of the first two and the last
two RCI values to obtain smoothed values of RCI for the N-terminal and
C-terminal residues, respectively.
19| Predict values of root
mean square fluctuations of MD ensembles (MD RMSF), mean square fluctuations of
NMR ensembles (NMR RMSF) and model-free order parameters (S2) 15,16 using the following expressions.
S2
= 1 - 0.5 ln (1 + RCI * 10.0) (2)
RMSF (MD) = RCI * 23.6 Å (3)
RMSF (NMR) = RCI * 12.7 Å (4)
CRITICAL STEP While these relationships are based on the
strong correlation between RCI and motional amplitudes 12,
we would like to stress the fact that these expressions are empirical. When
interpreting RCI in terms of MD RMSF, NMR RMSD, and S2,
one should realize that the motions described by these parameters may have
quite different time-scales. S2 and
MD RMSF represent protein dynamics on ps-ns
time-scale while RCI is expected to be mostly affected by the fast chemical
exchange from ps to ms-ms
(the coalescence points of nuclei involved in RCI calculations). More precise identification of motional time-scales using the
RCI method is not possible. The time-scale of NMR ensembles is hard to
estimate. The structural distribution in NMR ensembles often originates from
enhanced conformational sampling at the high-temperature steps of MD-based
structure calculation protocols and heavily depends on the balance between NMR
restraints and contributions of non-restraint terms of NMR force-fields. Regions
of NMR ensembles calculated with small number of restraints may demonstrate
unrealistic structural diversity and, as a result, low agreement with RCI due
to simplifications in non-bonded energy terms of NMR force-field. However, the
high correlation observed between RCI and aforementioned motional amplitudes 12
suggests that, despite the possible differences in the time-scales, these
parameters describe dynamics phenomena with a common determinant (e.g. protein
atomic density) and agree in identifying “hot” kinetic spots in protein
structures.
TIMELINE
When backbone
assignments are available, chemical shifts can be re-referenced and protein
flexibility can be predicted from the RCI protocol using a spreadsheet program within
1-2 hours. Users may also code the protocol
into a spreadsheet program so that subsequent analyses for new proteins can be
performed in just seconds.
ANTICIPATED RESULTS
RCI correlates
with the conventional measures of protein with an average of correlation
coefficient of ~ 0.8 12 (Table 3). Figure
1 shows examples of predicted MD RMSF, NMR RMSF and S2
and their comparison with values obtained by standard methods.
Table 3: Mean coefficients of
correlation between RCI and conventional measures of motional amplitudes 12.
Parameter |
# of proteins |
# of residues
b |
Correlation c |
MD RMSF |
18 |
2187 |
0.82 |
NMR RMSF |
17 |
2-15 |
0.81 |
Order parameter a |
18 |
2187 |
0.77 |
a order parameters predicted with a
contact model [Zhang, 2002 #1449 were used
whenever experimentally obtained order parameters were not available. b number
of residues in PDB files. c mean
correlation coefficient.
More detailed correlation statistics and 19 additional
plots with examples of excellent correlation between RCI and these parameters
can be found in the first report about the RCI method [Berjanskii,
2005 #1529].
In general, it is
not necessary to convert RCI to MD RMSF, NMR RMSF, and S2 to
effectively identify regions with elevated mobility in proteins. The
information content of RCI and the quality of the flexibility predictions does
not change upon such a conversion. However, RCI-derived traditional measures of
motional amplitudes could be easier to relate to models of protein motions and
compare with corresponding parameters obtained with standard methods. In such
cases, aforementioned equations 2, 3 and 4 can be used.
REFERENCES.
1. Carugo, O.
& Argos, P. Reliability of atomic displacement parameters in protein
crystal structures. Acta Crystallogr D Biol Crystallogr 55 ( Pt 2),
473-8 (1999).
2. Petsko, G. A. & Ringe, D.
Fluctuations in protein structure from X-ray diffraction. Annu Rev Biophys
Bioeng 13,
331-71 (1984).
3. Hansson,
T., Oostenbrink, C. & van Gunsteren,
W. Molecular dynamics simulations. Curr Opin Struct Biol 12, 190-6 (2002).
4. Elofsson, A. & Nilsson, L. How Consistent Are
Molecular-Dynamics Simulations - Comparing Structure and Dynamics in Reduced
and Oxidized Escherichia-Coli Thioredoxin. Journal of Molecular Biology
233, 766-780 (1993).
5. Kay,
L. E. Protein dynamics from NMR. Nat Struct Biol
5 Suppl,
513-7 (1998).
6. Ishima, R. & Torchia, D. A.
Protein dynamics from NMR. Nat Struct Biol
7, 740-3 (2000).
7. Palmer,
A. G., 3rd. Nmr probes of
molecular dynamics: overview and comparison with other techniques. Annu Rev Biophys
Biomol Struct 30, 129-55 (2001).
8. Lacroix, E., Bruix, M.,
Lopez-Hernandez, E., Serrano, L. & Rico, M. Amide hydrogen exchange and
internal dynamics in the chemotactic protein CheY from Escherichia coli. J Mol Biol 271, 472-87 (1997).
9. Korzhnev, D. M., Orekhov, V. Y.
& Arseniev, A. S. Model-free approach beyond the
borders of its applicability. J Magn Reson 127, 184-91 (1997).
10. Palmer,
A. G., 3rd, Kroenke, C. D. & Loria,
J. P. Nuclear magnetic resonance methods for quantifying
microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol
339, 204-38 (2001).
11. Fushman, D., Cahill, S. & Cowburn,
D. The Main-Chain Dynamics of the Dynamin Pleckstrin Homology (Ph) Domain in Solution - Analysis of
N-15 Relaxation With Monomer/Dimer
Equilibration. Journal
of Molecular Biology 266,
173-194 (1997).
12. Berjanskii,
M. V. & Wishart, D. S. A simple
method to predict protein flexibility using secondary chemical shifts. J Am Chem Soc 127, 14970-1 (2005).
13. Wishart, D. S., Sykes, B. D. & Richards, F. M. The
chemical shift index: a fast and simple method for the assignment of protein
secondary structure through NMR spectroscopy. Biochemistry 31, 1647-51 (1992).
14. Wishart, D. S. & Sykes, B. D. The 13C chemical-shift
index: a simple method for the identification of protein secondary structure
using 13C chemical-shift data. J Biomol NMR 4, 171-80 (1994).
15. Lipari,
G. & Szabo, A. Model-Free Approach to the
Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 1.
Theory and Range of Validity. Journal of the American Chemical Society 104, 4546-4559 (1982).
16. Clore, G. M. et al. Deviations from the simple
two-parameter model-free approach to the interpretation of nitrogen-15 nuclear
magnetic relaxation of proteins. Journal of the American Chemical Society 112, 4989-4991 (1990).
17. Markley,
J. L. et al. Recommendations for the presentation of NMR structures of proteins
and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group
on the Standardization of Data Bases of Protein and Nucleic Acid Structures
Determined by NMR Spectroscopy. J Biomol NMR 12, 1-23 (1998).
18. Wishart, D. S. et al. 1H, 13C and 15N chemical shift
referencing in biomolecular NMR. J Biomol NMR 6, 135-40 (1995).
19. Zhang,
H., Neal, S. & Wishart, D. S. RefDB:
a database of uniformly referenced protein chemical shifts. J Biomol NMR 25, 173-95 (2003).
20. Schwarzinger, S., Kroon, G. J.,
Foss, T. R., Wright, P. E. & Dyson, H. J. Random coil chemical shifts in
acidic 8 M urea: implementation of random coil shift data in NMRView. J Biomol NMR 18, 43-8. (2000).
21. Schwarzinger, S. et al. Sequence-dependent correction of
random coil NMR chemical shifts. Journal of the American Chemical Society 123, 2970-2978 (2001).
22. Wang,
Y. & Jardetzky, O. Probability-based protein
secondary structure identification using combined NMR chemical-shift data. Protein Sci
11, 852-61 (2002).
23. Iwahara, J., Peterson, R. D. & Clubb,
R. T. Compensating increases in protein backbone flexibility occur when the
Dead ringer AT-rich interaction domain (ARID) binds DNA: a nitrogen-15
relaxation study. Protein
Sci 14,
1140-50 (2005).
24. Zhang,
F. & Bruschweiler, R. Contact model for the
prediction of NMR N-H order parameters in globular proteins. J Am Chem Soc 124, 12654-5 (2002).
Supplementary
table 1. Random coil reference chemical shifts 20.
aa |
N |
CO |
Ca |
Cb |
NH |
Ha |
A |
125 |
178.5 |
52.8 |
19.3 |
8.35 |
4.35 |
C |
118.7 |
175.5 |
55.6 |
41.2 |
8.54 |
4.76 |
B |
118.8 |
175.3 |
58.6 |
28.3 |
8.44 |
4.59 |
D |
119.1 |
175.9 |
53 |
38.3 |
8.56 |
4.82 |
E |
120.2 |
176.8 |
56.1 |
29.9 |
8.4 |
4.42 |
F |
120.7 |
176.6 |
58.1 |
39.8 |
8.31 |
4.65 |
G |
107.5 |
174.9 |
45.4 |
- |
8.41 |
4.02 |
H |
118.1 |
175.1 |
55.4 |
29.1 |
8.56 |
4.79 |
I |
120.4 |
177.1 |
61.6 |
38.9 |
8.17 |
4.21 |
K |
121.6 |
177.4 |
56.7 |
33.2 |
8.36 |
4.36 |
L |
122.4 |
178.2 |
55.5 |
42.5 |
8.28 |
4.38 |
M |
120.3 |
177.1 |
55.8 |
32.9 |
8.42 |
4.52 |
N |
119 |
176.1 |
53.3 |
39.1 |
8.51 |
4.79 |
P |
- |
177.8 |
63.7 |
32.2 |
- |
4.45 |
Q |
120.5 |
176.8 |
56.2 |
29.5 |
8.44 |
4.38 |
R |
121.2 |
177.1 |
56.5 |
30.9 |
8.39 |
4.38 |
S |
115.5 |
175.4 |
58.7 |
64.1 |
8.43 |
4.51 |
T |
112 |
175.6 |
62 |
70 |
8.25 |
4.43 |
V |
119.3 |
177 |
62.6 |
31.8 |
8.16 |
4.16 |
W |
122.1 |
177.1 |
57.6 |
29.8 |
8.22 |
4.7 |
Y |
120.9 |
176.7 |
58.3 |
38.9 |
8.26 |
4.58 |
aa – amino acid one-letter code. B – reduced cysteine.
Supplementary table 2. Neighboring
residue corrections 21.
Part A: i+1 and i-1 corrections.
aa |
i-1 |
i+1 |
||||||||||
|
15N |
13C’ |
13Ca |
13Cb |
1HN |
1Ha |
15N |
13C’ |
13Ca |
13Cb |
1HN |
1Ha |
A |
-0.57 |
-0.07 |
0.06 |
0 |
0.07 |
-0.03 |
-0.33 |
-0.77 |
-0.17 |
0 |
-0.05 |
-0.03 |
R |
1.62 |
-0.19 |
-0.01 |
0 |
0.15 |
-0.02 |
-0.14 |
-0.49 |
-0.07 |
0 |
-0.02 |
-0.02 |
N |
0.87 |
-0.1 |
0.23 |
0 |
0.13 |
-0.02 |
-0.26 |
-0.66 |
-0.03 |
0 |
-0.03 |
-0.01 |
D |
0.86 |
-0.13 |
0.25 |
0 |
0.14 |
-0.02 |
-0.2 |
-0.58 |
0 |
0 |
-0.03 |
-0.01 |
C a |
3.07 |
-0.28 |
0.1 |
0 |
0.2 |
0 |
-0.26 |
-0.51 |
-0.07 |
0 |
-0.02 |
0.02 |
Q |
1.62 |
-0.18 |
0.04 |
0 |
0.15 |
-0.01 |
-0.14 |
-0.48 |
-0.06 |
0 |
-0.02 |
-0.02 |
E |
1.51 |
-0.2 |
0.05 |
0 |
0.15 |
-0.02 |
-0.2 |
-0.48 |
-0.08 |
0 |
-0.03 |
-0.02 |
G |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
H |
1.68 |
-0.22 |
0.02 |
0 |
0.2 |
0.01 |
-0.55 |
-0.65 |
-0.09 |
0 |
-0.04 |
-0.06 |
I |
4.87 |
-0.18 |
-0.01 |
0 |
0.17 |
-0.02 |
-0.14 |
-0.58 |
0.2 |
0 |
-0.06 |
-0.02 |
L |
1.05 |
-0.13 |
0.03 |
0 |
0.14 |
-0.05 |
-0.14 |
-0.5 |
-0.1 |
0 |
-0.03 |
-0.03 |
K |
1.57 |
-0.18 |
-0.02 |
0 |
0.14 |
-0.01 |
-0.2 |
-0.5 |
-0.11 |
0 |
-0.03 |
-0.02 |
M |
1.57 |
-0.18 |
-0.06 |
0 |
0.14 |
-0.01 |
-0.2 |
-0.41 |
0.1 |
0 |
-0.02 |
-0.01 |
F |
2.78 |
-0.25 |
0.06 |
0 |
0.15 |
-0.08 |
-0.49 |
-0.83 |
-0.23 |
0 |
-0.12 |
-0.09 |
P |
0.87 |
-0.09 |
0.02 |
0 |
0.1 |
-0.03 |
-0.32 |
-2.84 |
-2 |
0 |
-0.18 |
0.11 |
S |
2.55 |
-0.15 |
0.13 |
0 |
0.19 |
0 |
-0.03 |
-0.4 |
-0.08 |
0 |
-0.03 |
0.02 |
T |
2.78 |
-0.13 |
0.12 |
0 |
0.14 |
0 |
-0.03 |
-0.19 |
-0.04 |
0 |
0 |
0.05 |
W |
3.19 |
-0.3 |
0.03 |
0 |
0.04 |
-0.15 |
-0.26 |
-0.85 |
-0.17 |
0 |
-0.13 |
-0.1 |
Y |
3.01 |
-0.24 |
0.06 |
0 |
0.09 |
-0.08 |
-0.43 |
-0.85 |
-0.22 |
0 |
-0.11 |
-0.1 |
V |
4.34 |
-0.18 |
-0.02 |
0 |
0.17 |
-0.02 |
-0.14 |
-0.57 |
-0.21 |
0 |
-0.05 |
-0.01 |
Part B: i+2 and i-2 corrections.
|
i-2 |
i+2 |
||||||||||
aa |
15N |
13C’ |
13Ca |
13Cb |
1HN |
1Ha |
15N |
13C’ |
13Ca |
13Cb |
1HN |
1Ha |
A |
-0.15 |
-0.02 |
0.01 |
0 |
-0.1 |
0 |
-0.12 |
-0.11 |
-0.02 |
0 |
-0.01 |
-0.02 |
R |
-0.06 |
-0.03 |
0.02 |
0 |
-0.06 |
0 |
-0.06 |
-0.06 |
0 |
0 |
0 |
-0.02 |
N |
-0.17 |
-0.03 |
0.01 |
0 |
-0.07 |
-0.01 |
-0.18 |
-0.09 |
-0.06 |
0 |
-0.01 |
-0.01 |
D |
-0.29 |
-0.04 |
-0.01 |
0 |
-0.11 |
-0.01 |
-0.12 |
-0.08 |
-0.03 |
0 |
-0.02 |
-0.02 |
C a |
0 |
-0.07 |
-0.01 |
0 |
-0.07 |
0 |
-0.06 |
-0.08 |
-0.03 |
0 |
0 |
-0.01 |
B |
0 |
-0.07 |
-0.01 |
0 |
-0.07 |
0 |
-0.06 |
-0.08 |
-0.03 |
0 |
0 |
-0.01 |
Q |
-0.06 |
-0.03 |
0.01 |
0 |
-0.06 |
0 |
-0.06 |
-0.05 |
-0.02 |
0 |
-0.01 |
-0.01 |
E |
-0.12 |
-0.03 |
0.01 |
0 |
-0.07 |
0 |
-0.06 |
-0.09 |
-0.01 |
0 |
-0.01 |
-0.02 |
G |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
H |
0.17 |
-0.07 |
0.01 |
0 |
0 |
0.01 |
-0.12 |
-0.1 |
-0.05 |
0 |
-0.01 |
-0.03 |
I |
0 |
-0.02 |
0.02 |
0 |
-0.09 |
-0.01 |
-0.18 |
-0.2 |
-0.07 |
0 |
-0.01 |
-0.03 |
L |
-0.06 |
-0.01 |
0.02 |
0 |
-0.08 |
-0.01 |
-0.06 |
-0.13 |
-0.01 |
0 |
0 |
-0.04 |
K |
-0.06 |
-0.03 |
0.02 |
0 |
-0.06 |
0 |
-0.06 |
-0.08 |
-0.01 |
0 |
0 |
-0.02 |
M |
-0.06 |
-0.02 |
0.01 |
0 |
-0.06 |
0 |
-0.06 |
-0.08 |
0 |
0 |
0 |
-0.02 |
F |
-0.46 |
-0.1 |
0.01 |
0 |
-0.37 |
-0.04 |
-0.18 |
-0.27 |
-0.07 |
0 |
-0.03 |
-0.06 |
P |
-0.17 |
-0.02 |
0.04 |
0 |
-0.12 |
-0.01 |
-0.18 |
-0.47 |
-0.22 |
0 |
-0.04 |
-0.01 |
S |
-0.17 |
-0.06 |
0 |
0 |
-0.08 |
-0.01 |
-0.06 |
-0.08 |
0 |
0 |
0 |
-0.01 |
T |
-0.12 |
-0.05 |
0 |
0 |
-0.06 |
-0.01 |
-0.06 |
-0.08 |
-0.01 |
0 |
0.01 |
-0.01 |
W |
-0.64 |
-0.17 |
-0.08 |
0 |
-0.62 |
-0.16 |
0 |
-0.26 |
-0.02 |
0 |
-0.08 |
-0.08 |
Y |
-0.52 |
-0.13 |
-0.01 |
0 |
-0.42 |
-0.04 |
-0.24 |
-0.28 |
-0.07 |
0 |
-0.04 |
-0.05 |
V |
-0.06 |
-0.03 |
0.01 |
0 |
-0.08 |
-0.01 |
-0.24 |
-0.2 |
-0.07 |
0 |
-0.01 |
-0.02 |
a The same correction
factors are used for adjacent cysteine residues in
reduced and oxidized forms.
Supplementary table 3. Averaged
chemical shift (in ppm) observed in b-strands and a-helices
22.
|
13Ca |
13Cb |
13C’ |
1HN |
1Ha |
15N |
||||||
aa |
b |
a |
b |
a |
b |
a |
b |
a |
b |
a |
b |
a |
A |
50.86 |
54.86 |
21.72 |
18.27 |
175.3 |
179.58 |
8.59 |
7.99 |
4.87 |
4.03 |
125.57 |
121.65 |
R |
54.63 |
59.05 |
32.36 |
30 |
175.04 |
178.11 |
8.57 |
8.03 |
4.85 |
4 |
122.6 |
118.99 |
N |
52.48 |
55.67 |
40.43 |
38.28 |
174.55 |
176.74 |
8.7 |
8.2 |
5.26 |
4.45 |
122.7 |
117.6 |
D |
53.4 |
57.04 |
42.78 |
40.5 |
175.15 |
178.07 |
8.56 |
8.05 |
5.01 |
4.44 |
123.82 |
119.9 |
Q |
54.33 |
58.61 |
31.92 |
28.33 |
174.5 |
178.35 |
8.51 |
8.11 |
4.97 |
4.03 |
123.14 |
118.59 |
E |
55.55 |
59.3 |
32.45 |
29.2 |
175.01 |
178.46 |
8.66 |
8.32 |
4.76 |
3.99 |
123.52 |
119.89 |
G |
45.08 |
47.02 |
- |
- |
173.01 |
176.31 |
8.27 |
8.23 |
4.09 |
3.84 |
110.19 |
107.34 |
H |
54.8 |
59.62 |
32.2 |
29.91 |
173.8 |
176.83 |
8.76 |
8.03 |
5.07 |
4.06 |
121.65 |
118.09 |
I |
60 |
64.68 |
40.09 |
37.59 |
174.79 |
177.49 |
8.74 |
8.06 |
4.72 |
3.66 |
124.12 |
120.22 |
L |
53.94 |
57.54 |
44.02 |
41.4 |
175.16 |
178.42 |
8.63 |
8.02 |
4.85 |
4 |
125.69 |
120.18 |
K |
55.01 |
59.11 |
34.86 |
32.31 |
174.93 |
177.79 |
8.54 |
8.04 |
4.96 |
3.98 |
123.29 |
119.9 |
M |
54.1 |
58.45 |
34.34 |
31.7 |
174.64 |
177.76 |
8.43 |
8.05 |
4.94 |
4.03 |
121.67 |
118.69 |
F |
56.33 |
60.74 |
41.64 |
38.91 |
174.15 |
176.42 |
8.8 |
8.21 |
5.1 |
4.11 |
121.95 |
119.12 |
P |
62.79 |
65.52 |
32.45 |
31.08 |
176.41 |
178.34 |
- |
- |
4.72 |
4.13 |
- |
- |
S |
57.14 |
60.86 |
65.39 |
62.81 |
173.52 |
176.51 |
8.57 |
8.11 |
5.08 |
4.2 |
117.44 |
114.78 |
T |
61.1 |
65.89 |
70.82 |
68.64 |
173.47 |
176.62 |
8.5 |
8.1 |
4.81 |
4.02 |
118.09 |
115.3 |
W |
56.28 |
60.03 |
31.78 |
28.74 |
175.1 |
177.81 |
8.83 |
8.24 |
5.24 |
4.35 |
124.04 |
120.48 |
Y |
56.56 |
61.07 |
40.79 |
38.38 |
174.65 |
177.05 |
8.69 |
8.1 |
5 |
4.14 |
122.55 |
119.67 |
V |
60.72 |
65.96 |
33.81 |
31.41 |
174.66 |
177.75 |
8.73 |
7.99 |
4.66 |
3.5 |
123.27 |
119.53 |
B |
57.64 |
62.86 |
29.48 |
26.99 |
173.86 |
177.42 |
9 |
8.22 |
5.18 |
4.16 |
123.27 |
117.4 |
C |
54.19 |
58.57 |
43.79 |
40.02 |
172.73 |
176.84 |
8.68 |
8.58 |
5.21 |
4.53 |
121.81 |
119.51 |
aa – amino acid one-letter code. B
– reduced cysteine. b - beta-strand. a - a-helix
Supplementary Table 4. Weighting
coefficients for RCI calculation (equation 1) with different sets of chemical
shifts.
Nuclei included |
Weighting coefficients |
|||||
Ca |
CO |
Cb |
N |
Ha |
NH |
|
Ca, Cb, CO, N, Ha, NH |
0.74 |
0.72 |
0.13 |
0.38 |
0.91 |
0.15 |
Ca, Cb, CO, Ha, NH |
0.72 |
0.68 |
0.1 |
0 |
0.91 |
0.24 |
Ca, Cb, CO, N, NH |
0.85 |
0.82 |
0.35 |
0.32 |
0 |
0.21 |
Ca, Cb, N, Ha, NH |
0.8 |
0 |
0.16 |
0.34 |
0.88 |
0.09 |
Ca, CO, N, Ha, NH |
0.68 |
0.68 |
0 |
0.33 |
0.89 |
0.13 |
Cb, CO, N, Ha, NH |
0 |
0.79 |
0.05 |
0.33 |
0.89 |
0.08 |
Ca, Cb, CO, N, Ha |
0.71 |
0.67 |
0.13 |
0.43 |
0.88 |
0 |
Ca, Cb, CO, Ha |
0.64 |
0.6 |
0.1 |
0 |
0.82 |
0 |
Ca, Cb, CO, NH |
0.81 |
0.78 |
0.29 |
0 |
0 |
0.27 |
Ca, Cb, CO, N |
0.82 |
0.8 |
0.39 |
0.4 |
0 |
0 |
Ca, Cb, Ha, NH |
0.77 |
0 |
0.13 |
0 |
0.87 |
0.15 |
Ca, Cb, N, Ha |
0.77 |
0 |
0.16 |
0.36 |
0.84 |
0 |
Ca, Cb, N, NH |
0.86 |
0 |
0.37 |
0.28 |
0 |
0.11 |
Ca, CO, Ha, NH |
0.68 |
0.67 |
0 |
0 |
0.9 |
0.23 |
Ca, CO, N, Ha |
0.64 |
0.62 |
0 |
0.38 |
0.85 |
0 |
Ca, CO, N, NH |
0.82 |
0.79 |
0 |
0.22 |
0 |
0.28 |
Ca, N, Ha, NH |
0.75 |
0 |
0 |
0.3 |
0.87 |
0.1 |
Cb, CO, Ha, NH |
0 |
0.71 |
0.03 |
0 |
0.82 |
0.14 |
Cb, CO, N, Ha |
0 |
0.76 |
0.04 |
0.35 |
0.85 |
0 |
Cb, CO, N, NH |
0 |
0.96 |
0.28 |
0.28 |
0 |
0.2 |
Cb, N, Ha, NH |
0 |
0 |
0 |
0.27 |
0.77 |
0 |
CO, N, Ha, NH |
0 |
0.75 |
0 |
0.3 |
0.86 |
0.07 |
CO, N, Ha |
0 |
0.71 |
0 |
0.32 |
0.83 |
0 |
CO, N, NH |
0 |
0.93 |
0 |
0.27 |
0 |
0.2 |
N, Ha, NH |
0 |
0 |
0 |
0.27 |
0.77 |
0 |
Cb, N, Ha |
0 |
0 |
0 |
0.27 |
0.77 |
0 |
Cb, N, NH |
0 |
0 |
0.73 |
0.78 |
0 |
0.25 |
CO, Ha, NH |
0 |
0.67 |
0 |
0 |
0.78 |
0.13 |
Cb, CO, N |
0 |
0.84 |
0.28 |
0.32 |
0 |
0 |
Cb, Ha, NH |
0 |
0 |
0 |
0 |
0.6 |
0 |
Cb, CO, Ha |
0 |
0.61 |
0.05 |
0 |
0.76 |
0 |
Cb, CO, NH |
0 |
0.85 |
0.25 |
0 |
0 |
0.2 |
Ca, N, Ha |
0.71 |
0 |
0 |
0.32 |
0.83 |
0 |
Ca, N, NH |
0.83 |
0 |
0 |
0.17 |
0 |
0.23 |
Ca, CO, N |
0.78 |
0.76 |
0 |
0.35 |
0 |
0 |
Ca, Ha, NH |
0.71 |
0 |
0 |
0 |
0.85 |
0.13 |
Ca, CO, Ha |
0.56 |
0.56 |
0 |
0 |
0.78 |
0 |
Ca, CO, NH |
0.78 |
0.76 |
0 |
0 |
0 |
0.3 |
Ca, Cb, N |
0.83 |
0 |
0.38 |
0.32 |
0 |
0 |
Ca, Cb, Ha |
0.65 |
0 |
0.12 |
0 |
0.76 |
0 |
Ca, Cb, NH |
0.89 |
0 |
0.34 |
0 |
0 |
0.23 |
Ca, Cb, CO |
0.79 |
0.74 |
0.31 |
0 |
0 |
0 |
N, NH |
0 |
0 |
0 |
0.71 |
0 |
0.42 |
N, Ha |
0 |
0 |
0 |
0.27 |
0.77 |
0 |
CO, N |
0 |
0.77 |
0 |
0.27 |
0 |
0 |
Ha, NH |
0 |
0 |
0 |
0 |
0.6 |
0 |
CO, Ha |
0 |
0.55 |
0 |
0 |
0.69 |
0 |
CO, NH |
0 |
0.8 |
0 |
0 |
0 |
0.2 |
Cb, N |
0 |
0 |
0.65 |
0.68 |
0 |
0 |
Cb, Ha |
0 |
0 |
0 |
0 |
0.6 |
0 |
Cb, NH |
0 |
0 |
0.71 |
0 |
0 |
0.51 |
Cb, CO |
0 |
0.85 |
0.25 |
0 |
0 |
0 |
Ca, N |
0.77 |
0 |
0 |
0.27 |
0 |
0 |
Ca, Ha |
0.55 |
0 |
0 |
0 |
0.69 |
0 |
Ca, NH |
0.85 |
0 |
0 |
0 |
0 |
0.25 |
Ca, CO |
0.68 |
0.65 |
0 |
0 |
0 |
0 |
Ca, Cb |
0.74 |
0 |
0.34 |
0 |
0 |
0 |
Ca |
1 |
0 |
0 |
0 |
0 |
0 |
CO |
0 |
1 |
0 |
0 |
0 |
0 |
N |
0 |
0 |
0 |
1 |
0 |
0 |
Cb |
0 |
0 |
1 |
0 |
0 |
0 |
NH |
0 |
0 |
0 |
0 |
0 |
1 |
Ha |
0 |
0 |
0 |
0 |
1 |
0 |