1
Solid State NMR Study and Assignments of the KcsA
Potassium Ion Channel of S. lividans
Krisztina Varga[a], Lin Tian[a], Joshua A. Wand[b], Ann E. McDermott[a]*
Contributed from the Department of Chemistry, Columbia University, New York, NY
10027, Department of Biochemistry and Biophysics, University of Pennsylvania,
Philadelphia, PA, 19104
* To whom correspondence should be addressed: Columbia University, Department of
Chemistry, 3000 Broadway, New York, NY 10027, Phone: (212) 854-xxxx, email:
aem5@columbia.edu
[a] Columbia University
[b] University of Pennsylvania
Abstract: We carried out solid state NMR (SSNMR) studies leading site-specific
assignments of uniformly 13C, 15N enriched microcrystalline KcsA potassium channel
(160 residues) from Streptomyces lividans, an integral membrane protein. The
experimental strategy included a combination of 2D and 3D solid state NMR techniques
at 600 and 750 MHz. For most amino acids, signal patterns were established in 2D 13C13
C experiments. Since the secondary structure is non-helical in this region of the
protein, the chemical shifts of some residues are shifted from the typical helical residues
and provide a useful fingerprint for the sample. In the 3D spectra, residue types were
confirmed using side chain assignments. Assignments include both backbone and some
side chains atoms near and at the selectivity filter region of the protein. Potassium
channels are widely studied for their very efficient conductivity and selectivity, and our
study opens the door for structure determination and mechanism of ion conduction by
SSNMR.
Introduction
The purpose of this work was to establish the feasibility of site-specific assignments
and further biophysical studies of KcsA potassium ion channel from Streptomyces
lividans (S. lividans), a 160 amino acid integral membrane protein. Potassium channels
are fascinating proteins present in both prokaryotes and eukaryotes. Two remarkable
features of these channels are the very high selectivity towards K+ ions and their rates of
conductivity which approach the diffusion limit.1 KcsA functions as a homotetramer
(70.4 kDa). The monomer is composed of two trans-membrane helices connected by an
extracellular loop including the pore helix. The pore region sequence is very conserved,
thus likely all K+ channels (both ligand- and voltage-gated) from different species
essentially have the same gating mechanism and pore structure 1-3. KcsA, isolated from
S. lividans, is an integral membrane protein with similar structure and ion permeability to
other K+ channels. It is the first K+ channel for which the 3D structure has been
determined by X-ray diffraction to 3.2 Å resolution1 by MacKinnon and colleagues.
2
Later the resolution was refined to 2.0 Å for the channel-Fab complex.4 The crystals
used for both X-ray studies (PDB: 1BL81 and 1K4C4) were grown of a deletion version of
KcsA – both the N and C terminals were deleted.
The availability of X-ray structural information can facilitate NMR structural studies
and makes KcsA a good model system for other membrane protein studies. Conversely,
solid state NMR can offers advantages over other biophysical methods. One of the
advantages of solid state NMR studies is that X-ray quality crystals are not required –
microcrystalline protein precipitates are suitable for obtaining high resolution spectra.5
Thus it is feasible to study the functional, whole channel under various conditions,
including ligands, inhibitors, and toxins bound to it. NMR chemical shift changes report
on structural changes which may lead to a better understanding of the workings of
postassium channels. In this study KcsA was precipitated in detergent micelles, however
in subsequent studies the enzyme can be reconstituted into lipid bicelles, vesicles or as
2D crystals, which mimic the natural membrane environment even more closely. Solid
state NMR also offers the possibility to study protein dynamics of various resolved sites.
The prerequisite of structural studies of any protein by NMR is the assignment of
resonance peaks – the identification of which resonance peak corresponds to which
amino acid site specifically. KcsA is an integral membrane protein which exists mostly in
alpha-helical conformation (~50%), however other secondary structural elements are also
present6. There is high correlation between protein secondary structure and chemical
shifts 7-9. It is very difficult to assign all alpha helical proteins by NMR due to spectral
overlap. Beta sheet and other types of secondary structure elements facilitate
assignments because the chemical shift of those amino acids will be resolved from the
alpha-helical shifts. In KcsA, the functionally most interesting region, the pore
(containing the selectivity filter) is of mixed secondary elements thus was expected to be
most easily assignable. All K+ channels contain a critical, highly conserved amino acid
‘signature sequence’ 10, 11, which makes the selectivity filter of the channels and is
essential for proper function The signature sequence consists of the following amino
acids: T75, V76, G77, Y78, and G79 (in KcsA). To create a stack of main chain carbonyl
oxygen rings ligating the K+ ion, which is the structural basis of the channel selectivity,
the conformation of the five residues composing the selectivity filter alternates between
left- and right-handed alpha-helix, L and R respectively. The unique conformation
results in unusual backbone torsion angles, which yielded unusual shifts in the NMR
spectra for some of these residues. For instance, there were two valines easily
distinguishable from the other fourteen valine residues, which are likely part of the nonhelical pore region (Figure 2).
Understanding the three-dimensional structures and mechanisms of proteins
becomes increasingly important since it advances all the other areas of life sciences, such
as biology, pharmacology, and medicine. In the last few decades, the two major tools of
structural biology have been solution NMR and X-ray crystallography, and by the end of
2005 more than 30,000 soluble protein structures were solved to atomic resolution and
deposited in the Protein Data Bank (PDB), a web based single worldwide repository of
3D biological macromolecular structure data12. In contrast, integral membrane proteins
still pose a considerable challenge to biophysicists. Even though they constitute 20-30%
of the genome, only a handful membrane protein structures were determined (<2% of
known structures13) by these methods due to their low solubility and difficult
3
crystallization. Integral membrane proteins are involved in essential cell functions, such
as signal detection, import and export of nutrients and ions, cell-to-cell communication,
and energy production. Membrane proteins are a major part of drug receptors and a
better understanding of their structure and workings would lead to a significant
improvement in drug design and development. Recent technical advances in solid state
NMR (ssNMR) make it a very promising method for exploring membrane proteins and
other non-soluble or large biological structures that are difficult to study by solution
NMR and X-ray crystallography. Once the site specific assignments are obtained, the
sufficient number of structural constraints must be measured for 3D protein structure
determination. In the last few years, a large number of magic angle spinning (MAS)
multidimensional techniques developed at high fields which facilitated the assignment of
solid protein samples. Probably the first study that yielded spectra of high enough
resolution for site-specific assignments was of microcrystalline BPTI in 200014. Since
then, a few uniformly or extensively 13C, 15N enriched ssNMR soluble protein
assignments were published15-24. Membrane proteins present a bigger challenge than
soluble proteins, and to date only the LH2 light harvesting complex25, 26 has nearly
complete assignments. There are other membrane protein assignments in progress by
various research groups: Outer-membrane protein G27, Diaglycerol kinase28, and the LH1
light-harvesting complex29. The broader impact of the development of assignments
techniques in the solid state is that it would open new avenues to study protein structures
and dynamics. To date, a few small peptide30, 31 and protein (i.e. ubiquitin32, 33, -spectrin
SH3 domain34, and kaliotoxin35) 3D structures have been determined by solid state NMR.
KcsA is a well-suited sample for ssNMR studies was chosen as a model system for
several reasons: stability, availability, and biological importance. Long life time and
thermal stability make KcsA a very favorable target for ssNMR studies, since with the
currently available techniques the minimum experiment time is approximately a month
for collecting the 2D and 3D spectra necessary for assignment work. Sample heating is
another general consideration in MAS ssNMR experiments – samples heat up because of
the frictional forces due to spinning at high speed (~ 10-13 kHz). Even more importantly,
hydrated, high salt containing protein samples are also prone to heating caused by high
radio frequency (RF) power pulses, especially during long proton decoupling. KcsA is
stable at room temperature for extended periods of time in nonionic detergents, such as
DM6 and maintains the tetrameric form even at relatively high temperatures (up to
approximately 65 oC). For most membrane proteins the overexpression and purification
is still a challenge. KcsA can be expressed in E. coli and purification details have been
worked out 1.
Methods
Sample Preparation
Uniformly 13C, 15N enriched KcsA (~8.8 mg/ml) was overexpressed and purified
as previously described by (REF) mutations (Maybe brief description of purification??)
………..……………………………………………………………………………………
4
…….. U-13C, 15N KcsA from S. lividans was expressed in E. coli (strain??) in 4 mg
yield per liter of labeled minimal medium. After purification, protein purity was checked
by Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDSPAGE) according to the method of by Schagger and von Jagow 36. The protein bands
were visualized on a 10-20% gradient gel by silver staining. The protein sample was
stored at 4 ºC in 50 mM potassium phosphate buffer (pH 6.0), 50 mM KCl, 0.01 mM
sodium azide, 10 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM n-Dodecyl D-maltoside (DM). Protein concentration was determined by UV measurements at 280
nm ( 34,850 M-1 cm-1).
Approximately 2.3 mg of the protein was precipitated at room temperature by
mixing equal volumes of protein solution and precipitant (50% PEG 1000, 50 mM Tris,
100 mM KCl, pH 7.4). After precipitation the protein was stored at 4 oC. Ethylene
glycol and KCl were added to the solution to a final concentration of 15% (w/v) and 100
mM, respectively. The precipitate was then collected by centrifugation at 25,000 g
(16,400 rpm) and transferred to a 4 mm Bruker NMR rotor. 2D 13C-13C homonuclear
correlation spectra (DARR 37) were acquired at 750 MHz (Bruker Avance DRX-750
spectrometer). After data collection, approximately 1.2 mg of this sample was repacked
into a 4 mm Varian NMR rotor. The K+ concentration was adjusted to approximately 110
mM before repacking. 2D 13C-13C homonuclear correlation spectra (DARR) and 3D
inter-residue (NCOCX) and intra-residue (NCACX) spectra of the repacked sample were
acquired on a Varian Infinity Plus 600 spectrometer.
Solid State NMR: Pulse Sequences and Acquisition Parameters
The techniques and pulse sequences used are similar to those successfully applied
for the assignment of backbone and sidechains of ubiquitin 20, 38. The assignments of
KcsA are based on four spectra: two 2D 13C-13C homonuclear DARR spectrum acquired
at 750 MHz and 600 MHz and two 3D DCP-DARR spectra recorded at 600 MHz.
A cartoon diagram of the pulse sequences is shown in the Supplementary
Materials. 2D 13C-13C homonuclear correlation spectra of KcsA were acquired by the
DARR (Dipolar Assisted Rotational Resonance) 37 method. After the initial ramped
magnetization transfer 39 from 1H to 13C by cross polarization at the Hartmann-Hahn
condition, simple proton assisted spin-diffusion mediates homonuclear polarization
transfer. During the mixing time the proton field strength is matched with the rotational
frequency of the rotor. 3D NCACX and NCOCX spectra were acquired by the DCPDARR sequence 20, which combines a selective double cross polarization (DCP) 40, 41
sequence and DARR. The double cross-polarization sequence (DCP) was used to
selectively direct the polarization from 15N to 13CA or 13CO, which was then transferred
to other 13C nuclei by DARR.
Acquisition Parameters
2D 13C-13C homonuclear correlation spectra of KcsA were acquired by the DARR
37
method on a Bruker Avance DRX-750 spectrometer. The spectrometer was operating
at Larmor frequencies of 750.22 MHz for 1H and 188.660969 MHz for 13C. The
experiments were carried out at 250 K and at 10 kHz spinning frequency. Note that 250
K was the temperature of the air cooling the sample; the actual sample temperature was
higher due to frictional forces from magic angle spinning and RF pulses. The sample
5
heating is not well calibrated at this point; it is estimated to be approximately 20 K higher
than the apparent temperature.
H 67.6 kHz, C 45.5 kHz, 0.8 ms
Cp conditions ……………………………………….. There were 2600 points
collected in the direct and 1024 points in the indirect dimension. The FID’s were
acquired for 18.2 ms in the direct dimension and 12.8 ms in the indirect dimension. The
spectrum was collected for 28 hours (32 scans with 3 s pulse delay). During mixing the
proton field strength was matched to the spinning frequency. The mixing time was 13
ms, which was sufficient to allow the observation of 2-3 bond transfers for most amino
acids.
2D 13C-13C homonuclear correlation spectra were also collected on a Varian
Infinity Plus 600 spectrometer at 253K and a 13 kHz spinning frequency. The DARR
mixing time was increased to 200 ms in order to identify medium range contacts. Cross
polarization was achieved by a 67 kHz square 1H pulse at 599.26325 MHz and 48- 58
kHz tangent ramped 13C pulse at 150.70005 MHz for 1 ms. There were 1024 points
collected both in the direct and indirect 13C dimensions for 17 ms and 13 ms,
respectively. During acquisition 71 kHz TPPM decoupling pulse was applied on the 1H
channel. During the 200 ms mixing the proton field was matched to the spinning
frequency. There were 192 scans collected with 3 s pulse delay.
3D 13C/15N chemical shift correlation solid-state NMR spectra were acquired on a
Varian Infinity Plus 600 spectrometer with a spinning frequency of 13 kHz and an
apparent sample temperature of 243 K. The spectrometer was operating at Larmor
frequencies of 599.290000 MHz for 1H, 60.732297 MHz for 15N, and 150.717491 MHz
and 150.698970 MHz for 13CO and 13CA, respectively. Two 3D experiments were carried
out: an NCOCX experiment for inter-residue backbone-backbone and backbonesidechain correlations and an NCACX experiment for intra-residue correlations.
For the 3D NCOCX inter-residue correlation experiment, 1H-15N crosspolarization was carried out using a tangent radio frequency (RF) pulse on the 15N
channel, with RF field of 53.7 kHz for the 1H channel and 33.7 kHz (at the center of the
ramp, with 10 kHz ramp) for the 15N channel. The contact time was 0.7 ms. The 13CO
was selectively polarized by a 45.9 kHz 15N pulse and a 30.4 kHz 13C pulse with a slight
ramp of 2.0 kHz for 2.0 ms. Homonuclear 13C-13C mixing was accomplished using the
DARR pulse sequence with a mixing time of 19.23 ms. There were 1024 points collected
in the direct 13C dimension, 48 points in the indirect 15N dimension, and 32 points in the
indirect 13C dimension. Data were recorded for 16.89 ms in the direct dimension, 7.38 ms
in the indirect 15N dimension, and 4.96 ms in the indirect 13C dimension. The spectrum
was collected for 6.9 days, in 8 blocks with 20.8 hours duration for each block.
For the NCACX intra-residue correlation experiment, 1H-15N cross-polarization
was carried out using a tangent radio frequency field on the 15N channel, with the field
strength of 53.7 kHz for 1H and 34.5 kHz for 15N (at the center of the ramp), with a 10
kHz ramp for a duration of 0.7 ms. A ramped radio frequency field was used on the 13C
channel with the field strength of 28.8 kHz for the 15N and 17.1 kHz for the 13CA (at the
center of the ramp), ramp size of 1.4 kHz, and a contact time of 2.0 ms. Homonuclear
13 13
C- C mixing was accomplished using DARR sequence with a mixing time of 19.20
ms. There were 1024 points collected in the direct 13C dimension, 48 points in the
indirect 15N dimension, and 48 points in the indirect 13C dimension. The FIDs were
6
recorded for 16.89 ms in the direct dimension, 7.38 ms in the indirect 15N dimension, and
3.72 ms in the indirect 13C dimension. The spectrum was collected for 6.5 days, in 12
blocks with 13.0 hours duration for each block.
NMR Data Processing
All data were processed with NMRPipe 42; analysis and assignments of the 2D
and 3D data sets were carried out using Sparky version 3.1 43. All spectra were processed
at least two different ways: (i) by applying only sinebell apodization in each dimension;
(ii) by applying exponential multiplication in order to enhance the signal-to-noise, which
proved to be valuable for the assignment of weak isolated peaks.
Both dimensions of the 2D DARR spectra were apodized by sinebell window
function and zero filled to 4096 points. The spectra were also processed by applying
exponential line broadening in both the direct and indirect dimensions (50 and 100 Hz
respectively), then the FIDs were zero filled to 4096 points before Fourier transform.
Both the NCACX and NCOCX 3D spectra were processed with sinebell
apodization function in both the direct and indirect dimensions. Alternatively, the
NCACX 3D spectrum was also processed with exponential line broadening: 100 Hz in
the observed 13C dimension, 40 Hz and 100 Hz broadening in the indirect 15N and 13C
dimensions respectively. For the NCOCX 3D spectrum, 100 Hz, 20 Hz, and 50 Hz
exponential line broadening was applied in the observed 13C and indirect 15N and 13C
dimensions respectively. In each case the direct carbon dimension was zero filled to
4096 points, while the indirect 13C and 15N dimensions were both zero-filled to 256
points. For each experiment, the carbon dimension was referenced externally to TMS
using the 13C adamantane methylene peak at 38.56 ppm, which was then adjusted to DSS
assuming a 1.7 ppm chemical shift difference 44. The nitrogen dimension was referenced
to 15N labeled ammonium chloride…………..
Results and Discussion
There were two 2D 13C-13C DARR mixing spectra acquired: a DARR spectrum
with 13 ms mixing time which allowed for the observation of two bond transfers for most
amino acids and a spectrum with 200 ms mixing time for in order to observe medium
range contacts. The short mixing time 2D 13C-13C DARR mixing spectrum is usually the
first 2D spectrum recorded. These spectra are used to confirm residue types and the
secondary structure elements of the protein (i.e. helical nature). Since 13C chemical shifts
are sensitive to secondary structure changes, the 2D 13C-13C DARR mixing spectra were
also used to confirm sample integrity throughout the course of NMR measurements. As
reported by Zech et al.32, the majority of sequential contacts (<3.8 Å) can be observed at
200 ms. Due to the additional crosspeaks spectral crowding can be considerable,
therefore long mixing times are usually employed for proteins with selective labeling
(i.e. proteins purified from bacteria grown with 1,3-13C or 2-13C glycerol). For KcsA the
200 ms spectra were very congested for most regions as expected for a U-13C labeled 160
amino acid protein. However, some regions provided very useful insights into the
assignments.
As the initial step of the assignment, the amino acid type of most cross peak
clusters is identified according to their typical chemical shift and connectivity. Since
most of the protein has helical secondary structure, there are very congested areas. On
7
the other hand, there are some well resolved peaks in the spectrum either because there
are only a few residues of a certain type of amino acid, or because amino acids have a
secondary structure other than right handed alpha-helical. Because of the unique
chemical shifts, these ‘outliers’ can be considered as a ‘finger-print’ region and were used
to monitor sample integrity.
One example for sparse amino acid type is isoleucine, since the KcsA sequence
contains only three of them (I38, I60, I100). The C and the sidechain peaks for two
isoleucines can be clearly assigned in the 13 ms DARR spectrum, however the CO peaks
could not be assigned unambiguously. The third isoleucine was not observed under our
experimental conditions. In the 200 ms DARR spectrum provided additional, well
resolved CD-CO peaks which confirmed the CO assignments unambiguously, as
illustrated in the Figure xx in the Supplementary Materials. According to chemical shift
prediction by ShiftX45 based on the crystal structure of KcsA4 (PDB accession code:
1K4C), two of the three isoleucines should have chemical shifts which correspond to
alpha-helical confirmations, and I60 should have a chemical shifts which corresponds to
beta-sheet. Therefore, although not confirmed by sequential backbone walk, the
isoleucine with beta-sheet like chemical shift is most likely I60.
Other unique examples of outliers were found among the valines. Two valines
outliers could be readily identified in the DARR spectra according to their connectivity.
Since these valines have such different chemical shifts from the other 14 valines, it was a
likely hypothesis that these valines are part of the pore region. We pursued this
hypothesis and found plausible backbone walks in the 3D spectra. Based on the
backbone walk, we assigned the valine outliers as V76 and V84. Figure 2 illustrates the
two valine outliers in the DARR spectra, and Figure 4 shows the strip plots of the
backbone walk form the 3D experiments. Site-specific assignments for the Y82, P83,
and V84 carbonyls were also supported from the 200 ms DARR spectrum. The P83 CD
correlation with P83 CO, and the CO of the neighboring residues Y82 and V84 were
identified. The CO chemical shifts matched the assignments obtained from the 3D
spectra (Figure 5).
The backbone assignment strategy in the 3D spectra is very analogous to that used
in solution NMR. The polarization transfer pathway implemented by the 3D DCPDARR experiments is presented in the Supplementary Materials. In the NCACX
experiment, the polarization is selectively transferred from 15N to the 13CA of the same
residue by DCP, which is then distributed to the 13CO and also to the side chains by the
DARR sequence. Intra-residue correlations are obtained in this experiment.
The
NCOCX experiment is very similar, however the transfer is optimized for 15N to the 13CO
of the preceding residue, which is then distributed to the 13CA and the sidechains by
DARR. The NCOCX experiment provides inter-residue correlations thus making the
‘backbone walk’ feasible.
In the 3D spectra the 15N dimension dramatically improved the resolution for
some of the very congested regions of the 2D 13C-13C DARR spectrum. This is
illustrated in Figure 3 for the alanine CA-CB region. The KcsA sequence has 24
alanines, and the CA-CB crosspeak region can be readily identified in the DARR
spectrum (Figure 3A) based on their typical alpha-helical alanine chemical shifts (55.2
ppm for CA and 19.7 for CB 46). However, only one peak can be resolved in the DARR
spectrum. In the NCOCX 3D spectrum the congestion is relieved by the combination of
8
15
N chemical shift dispersion of the proceeding residues and the alanine 13CO dispersion.
Unique sidechain shifts can further improve resolution as illustrated in Figure 3B. Based
on the 15N-13CA-13CO chemical shifts only three alanines can be resolved, however the
CB shifts further improve the resolution and four peaks can be identified.
Sidechains also played an important role in confirming assignments for the
backbone walk. In Figure 4, strip plots of sequential assignments are displayed for some
residues where not only CA-CO backbone cross peaks but also the sidechains support the
identity of the specific amino acids. These side chains were also confirmed by the 2D
13 13
C- C DARR spectrum. Assignment efficiency was also limited by the particularly
weak peaks in regions with 15N chemical shift lower than 115 ppm or higher than 130
ppm. Usually these are the regions where outliers are found which often facilitate the
assignment process. For the identification of peaks in these regions (i.e. peaks with
glycine or proline 15N correlations), spectra processed with exponential line broadening
were used in almost each case.
In summary, sequential peak assignments were determined for residues V76-D80
and P83-C90 of KcsA. During the assignment process, peaks identified in the 3D spectra
were cross-checked against peaks observed in the 2D 13C-13C DARR spectrum. There
was a close agreement between the 2D and the 3D spectra. The backbone walk is
illustrated by the strip plots in Figure 4. The assigned regions are underlined in the KcsA
sequence in Figure 1. Both of these regions are part of the pore, and V76-D80 is also part
of the selectivity filter. The assigned regions are highlighted in the crystal structure of
KcsA (PDB: 1BL8) in the Supplementary Materials. Although these assignments need to
be confirmed by future experiments, there is a high probability that at least some of them
are correct due to the lack of supportive evidence for alternate lists.
Improving resolution in future experiments can facilitate assignments. Narrow
linewidths are especially important for mostly alpha-helical membrane proteins where
backbone carbons exhibit very congested spectra due to conformational similarity. 3D
pulse sequences implemented with homonulear 13C J decoupling (i.e. DANTE sequence)
during evolution in the indirect dimension could also significantly improve resolution, as
has been demonstrated for ubiquitin 47. Selective isotope labeling can also reduce
homonuclear dipolar couplings; the use of site directed biosynthetic labeling has been
demonstrated before to be useful for ssNMR assignment studies of the -spectrin SH3
domain 34 and for the most recent studies of LH2 26, 48: E. coli grown in minimal medium
supplemented with [2-13C] or [1,3-13C] glycerol produces proteins with parse labeling 49,
50
in a specific pattern that most labeled carbons have unlabeled neighbors.
This study forms the basis for other biophysical studies which could explore the
ion conduction. Solid state NMR studies do not require diffraction quality crystals,
therefore the precipitation conditions are greatly flexible for the protein. Since KcsA can
be precipitated under various conditions, structural changes induced by pH, salt
concentration or toxin binding can be easily detected by observing perturbed chemical
shifts.
Conclusions
9
The assignment of integral membrane proteins by solid state NMR is still a
difficult challenge. Our study forms the basis for a broader investigation for the structure
and mechanism of ion conduction of the KcsA potassium ion channel, an integral
membrane protein by solid state NMR. The prerequisite for NMR based structure
determination is the assignment of the observable 13C and 15N resonances to
corresponding nuclei. As the first step of the study, we established solid state NMR
sample preparation method for a homogenous sample. Solid state NMR sample
preparation method is crucial in obtaining narrow line widths thus resolving peaks, as has
been underlined by several studies (REF SH3 and ubiquitin)27, 28. The feasibility of the
sequential, site-specific assignments of KcsA backbone and sidechains is well
demonstrated in this study. The hypothetical assignments of the pore region and
selectivity filter were based on a combination of 2D and 3D NMR spectra acquired at
high field. This study demonstrates that solid-state NMR methods provide a promising
biophysical approach for many important and challenging biological systems.
10
11
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