A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data

Article abstract of DOI:10.1007/s10858-011-9545-x

The measurement of ¹H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.

Full text of DOI:10.1007/s10858-011-9545-x

J Biomol NMR (2011) 51:105–114
DOI 10.1007/s10858-011-9545-x
ARTICLE
A rigid disulfide-linked nitroxide side chain simplifies
the quantitative analysis of PRE data
•
Nicolas L. Fawzi Mark R. Fleissner
•
•
•
Nicholas J. Anthis Tamas Kalai Kalman Hideg
•
´
Wayne L. Hubbell G. Marius Clore
´
´
´
•
Received: 6 May 2011 / Accepted: 22 June 2011
Ó Springer Science+Business Media B.V. 2011
1
Abstract The measurement of H transverse paramag-
netic relaxation enhancement (PRE) has been used in
biomolecular systems to determine long-range distance
restraints and to visualize sparsely-populated transient
states. The intrinsic flexibility of most nitroxide and metal-
chelating paramagnetic spin-labels, however, complicates
the quantitative interpretation of PREs due to delocaliza-
tion of the paramagnetic center. Here, we present a novel,
disulfide-linked nitroxide spin label, R1p, as an alternative
to these flexible labels for PRE studies. When introduced at
solvent-exposed a-helical positions in two model proteins,
calmodulin (CaM) and T4 lysozyme (T4L), EPR mea-
surements show that the R1p side chain exhibits dramati-
cally reduced internal motion compared to the commonly
used R1 spin label (generated by reacting cysteine with
the spin labeling compound often referred to as MTSL).
Further, only a single nitroxide position is necessary to
account for the PREs arising from CaM S17R1p, while an
ensemble comprising multiple conformations is necessary
for those observed for CaM S17R1. Together, these
observations suggest that the nitroxide adopts a single,
fixed position when R1p is placed at solvent-exposed
a-helical positions, greatly simplifying the interpretation of
PRE data by removing the need to account for the intrinsic
flexibility of the spin label.
Keywords Paramagnetic relaxation enhancement ꢀ Site-
directed spin-labeling ꢀ Electron paramagnetic resonance
Introduction
In recent years, a number of research groups have devel-
oped a variety of NMR methods to probe biomolecular
structure and dynamics based on the site-specific intro-
duction of paramagnetic species (Clore 2008, 2011; Clore
and Iwahara 2009; Keizers and Ubbink 2011; Otting 2010).
The most generally applicable method for introducing
paramagnetic species is site-directed spin labeling (SDSL)
(Hubbell et al. 2000; Todd et al. 1989), in which a cysteine
is introduced at a site of interest by site-directed muta-
genesis, and then modified with a sulfhydryl-specific
compound to generate a spin label side chain. Due to the
large dipolar interaction between the unpaired electrons of
the paramagnetic species and the nuclear spins of the
biomolecule, position-specific distance, angular informa-
tion, or both can be extracted. Among the various para-
magnetic NMR techniques, the measurement of transverse
paramagnetic relaxation enhancements rates (PREs), par-
ticularly of 1HN nuclei, has shown broad applicability,
allowing a number of important questions in structure and
dynamics to be addressed (Fawzi et al. 2010; Iwahara
and Clore 2006; Iwahara et al. 2004a, 2006; Takayama and
Clore 2011; Tang et al. 2006, 2008, 2007; Volkov et al.
Nicolas L. Fawzi, Mark R. Fleissner and Nicholas J. Anthis
contributed equally.
N. L. Fawzi ꢀ N. J. Anthis ꢀ G. M. Clore (&)
Laboratory of Chemical Physics, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MA 20892-0520, USA
e-mail: mariusc@mail.nih.gov
M. R. Fleissner ꢀ W. L. Hubbell (&)
Jules Stein Eye Institute and Department of Chemistry
and Biochemistry, University of California, Los Angeles,
CA 90095, USA
e-mail: hubbellw@jsei.ucla.edu
´
T. Kalai ꢀ K. Hideg
Institute of Organic and Medicinal Chemistry,
´ ´
University of Pecs, Szigeti str. 12, 7624 Pecs, Hungary
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J Biomol NMR (2011) 51:105–114
nitroxide positions, their interconversion dynamics and
the order parameters for the interspin (nitroxide-nucleus)
vectors (Clore and Iwahara 2009; Iwahara et al. 2004b).
To simplify the interpretation of NMR PRE data, an
ideal spin label would not perturb the intrinsic structure
and dynamics of the protein, and would have a single
paramagnetic center that is rigidly attached to the protein.
Indeed, several relatively rigid spin labels have been
evaluated, but their general use for PRE studies appears to
be limited. One such label is TOAC, an amino acid
containing a stable nitroxide radical that can be intro-
duced into synthetic peptides and has been used suc-
cessfully for PRE studies (Lindfors et al. 2011). Although
both the Ca and the nitroxide of TOAC are contained
within a single piperidine ring, the radical can occupy at
least two distinct positions because of ring isomerizations
(Crisma et al. 2005; Flippen-Anderson et al. 1996).
Moreover, the utility of TOAC for PRE studies is limited
because its incorporation into large or isotope labeled
proteins is not practical, and also risks perturbing local
protein structure and dynamics. Two strategies, primarily
used for the measurement of pseudo-contact chemical
shifts (PCS), rely on multiple functional groups, either
engineered or existing, in order to introduce paramagnetic
ion chelating moieties. One is CLaNP-5 (Keizers et al.
2007), a large, organic molecule pre-chelated to a lan-
thanide ion (when cysteine bound, 614 Da plus a
139–175 Da lanthanide ion, compared to 185 Da for R1),
that can be introduced via two proximally located, surface
cysteine residues. The other is 4-mercaptomethyl-dipico-
linic acid (Su et al. 2008), which requires (i) a carboxyl
bearing side chain to be located near a single cysteine
residue for incorporation and (ii) the addition of lantha-
nide ions to the protein sample. This labeling strategy is
incompatible with metal-binding proteins, and may cause
protein aggregation due to non-specific protein-lanthanide
binding. Another recent strategy also designed for gen-
erating PCS is DOTA-M8, a stereospecifically methylated
DOTA lanthanide chelating label (Haussinger et al. 2009).
Although this group is relatively rigid and attachment
requires only a single cysteine, it is relatively large
(572 Da when cysteine bound, plus the lanthanide ion)
and hydrophobic due to methylation to increase protein
surface interactions, and the authors report that some
motion of the paramagnetic center with respect to the
backbone is evident by comparing the magnitude of the
PCS and RDC susceptibility tensors. The requirement for
multiple functional groups to incorporate the dipicolinic
acid and CLaNP-5 labels, can limit those strategies to
specific protein topologies while the large size of the
CLaNP and DOTA based labels may perturb the structure,
dynamics, and stability of the protein under investigation.
Thus, a nitroxide label that can be incorporated via a
A
Protein
O
S
S
O
S
S
+ Protein–SH
N
O
N
O
MTSL
R1 side chain
B
Protein
O
S
S
S
N
N
O
S
+ Protein–SH
N
O
N
O
HO-3606
R1p side chain
Fig. 1 Reaction of the widely used spin-labeling compound
(‘‘MTSL’’) and HO-3606 with a protein sulfhydryl group to generate
the R1 and R1p side chains, respectively
2010, 2006). PREs arising from site-directed introduction
of nitroxide spin labels or paramagnetic metal ions are
˚
large enough to reliably measure distances up to 35 A from
the unpaired electron and to probe transient, sparsely-
populated intra- or inter-molecular interactions that occa-
sionally bring the spin label position in close proximity to
1H nuclei of interest (Iwahara et al. 2004b; 2007).
Up to this point, however, the motion of the spin label
relative to the protein has complicated direct interpreta-
tion of both intra- and intermolecular PREs due to the
conformational space that can be sampled by the para-
magnetic center (Iwahara et al. 2004b). Among the
myriad spin labels available, R1 (Fig. 1a) is the most
widely used for EPR studies, and has also been used
successfully in several PRE studies (Bermejo et al. 2009;
Volkov et al. 2006). At solvent-exposed helical sites, the
R1 side chain occupies a set of rotamers that are limited
from the backbone through the disulfide linkage region
due in part to interactions of the disulfide with the peptide
backbone (Fleissner et al. 2009; Guo et al. 2008; 2007;
Warshaviak et al. 2011). Transitions between rotamers in
this region of the side chain change the spatial position of
the nitroxide relative to the protein on the time scale of
microseconds (Bridges et al. 2010), though additional
rotamer transitions for the terminal rotatable bonds are
site-dependent and on a much faster (likely in the ns
range) time scale (Chou et al. 2003). NMR studies
attempting to quantitatively interpret the PREs arising
from R1 (Bermejo et al. 2009), like any other label with
internal motions, must take into account the ensemble of
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J Biomol NMR (2011) 51:105–114
107
Synthesis of 3-Formyl-4-(pyridin-3-yl)-2,2,5,5-tetramethyl-
2,5-dihydro-1H-pyrrol-1-yloxyl radical [2]
single surface cysteine residue that combines the reliable,
sequence-context independent rigidity of a lanthanide-
based label with the ease of conjugation and small
chemical structure of R1, would be of greater general use
for PRE studies.
To a deoxygenated, stirred solution of aldehyde 1 (988 mg,
4.0 mmol) in dioxane (15 mL), Pd(PPh3)4 (100 mg,
0.1 mmol) was added in one portion and the mixture was
stirred at room temperature (r.t.) for 10 min. Next,
3-pyridineboronic acid (504 mg, 4.1 mmol) was added
followed by the addition of aqueous 10% Na2CO3 (10 mL,
9.4 mmol), and the mixture was stirred and refluxed for 2 h
under N2. After cooling, the solvents were evaporated off
in vacuo, and the residue was partitioned between CHCl3
(25 mL) and water (10 mL). The organic phase was sep-
arated, dried (MgSO4), filtered and evaporated to give
aldehyde 2. Yield: 617 mg (63%) after flash chromatog-
raphy purification (CHCl3/Et2O, 2:1), yellow solid, mp
98–99°C, Rf : 0.21 (CHCl3/Et2O, 2:1). MS EI m/z (%): 245
(M?, 100), 230(30), 200 (42), 172(88). IR (Nujol): 1670
(C=O), 1640, 1560, 1535 (C=C) cm-1. Composition cal-
culated for C14H17N2O2: C, 68.55; H, 6.99; N, 11.42.
Found: C, 68.50; H, 7.00; N, 11.36.
A suitable candidate may come from a series of R1
analogs, which show more restricted internal motions than
R1 at solvent-exposed sites (Columbus et al. 2001) and
presumably have fewer allowed conformations due to the
presence of a rigid substituent at position 4 of the pyrroline
ring. In particular, the 4-phenyl analog exhibits highly
restricted motions and adopts a single conformer at a model
solvent-exposed helical site (Fleissner 2007) (PDB code
1ZUR). However, spin labeled proteins bearing this par-
ticular derivative are prone to aggregation, probably due to
an increase in surface hydrophobicity. To circumvent this
problem, we synthesized a reagent (HO-3606) that gener-
ates a more hydrophilic, 4-pyridyl derivative of R1 (des-
ignated R1p, Fig. 1b) and evaluated its general use for PRE
studies.
First, we examined the effect of the pyridyl group on the
internal flexibility of the spin label side chain by EPR
spectroscopy by introducing it into solvent-exposed helical
sites in T4 lysozyme (T4L) and human calmodulin (CaM)
as model systems. We demonstrate that the EPR spectra of
R1p mutants of both proteins are consistent with a single,
highly ordered component, and, most importantly, that the
PREs arising from R1p can be accounted for by a single
label conformation, greatly simplifying the procedure for
using PREs as long range distance restraints or as probes of
transient states.
Synthesis of 3-Hydroxymethyl-4-(pyridin-3-yl)-2,2,5,5-
tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radical [3]
To a solution of aldehyde 2 (980 mg, 4.0 mmol) in EtOH
(15 mL), NaBH4 (190 mg, 5.0 mmol) was added in one
portion, and the mixture was stirred at r.t. for 15 min. and
then quenched with water (10 mL). The solution was
extracted with CHCl3 (2 9 10 mL), the organic phase was
dried (MgSO4), filtered and evaporated and the residue was
purified by flash column chromatography (CHCl3/MeOH,
9:1) to offer alcohol 3. Yield: 830 mg (84%), off-white
solid, mp 145-147°C, Rf : 0.14 (CHCl3/MeOH, 9:1). MS EI
m/z (%): 247 (M?, 100), 232(61), 217 (16), 184(35). IR
Experimental methods
(Nujol): 3150 (OH), 1620, 1560, 1535 (C=C) cm-1
.
Synthesis of the methanethiosulfonate
reagent HO-3606
Composition calculated for C14H19N2O2: C, 67.99; H,
7.74; N, 11.33. Found: C, 67.92; H, 7.71; N, 11.25.
The reagent HO-3606 (radical 5) was prepared according to
the general procedure of Scheme 1. Melting points were
determined with a Boetius micro melting point apparatus and
are uncorrected. Elemental analyses (C, H, N, S) were per-
formed on Fisons EA 1110 CHNS elemental analyzer. Mass
spectra were recorded on a Finnigan Automass-Multi
instrument in the EI mode. The IR (Specord 85) spectra were
in each case consistent with the assigned structure. Flash
column chromatography was performed on Merck Kieselgel
60 (0.040–0.063 mm). Qualitative TLC was carried out on
commercially prepared plates (20 9 20 9 0.02 cm) coated
with Merck Kieselgel GF254. Compound 1 was prepared
according to published procedures (Kalai et al. 1998);
3-pyridineboronic acid and other reagents were purchased
from Aldrich.
Synthesis of 3-Chloromethyl-4-(pyridin-3-yl)-2,2,5,5-
tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radical [4]
To a solution of alcohol 3 (1.23 g, 5.0 mmol) in CH2Cl2
(15 mL) and Et3N (555 mg, 5.5 mmol), MeSO2Cl
(630 mg, 5.5 mmol) was added at 0°C. The mixture was
stirred at r.t. for 30 min., then washed with water (10 mL),
and the organic phase was separated, dried (MgSO4), fil-
tered and evaporated. The residue was dissolved in dry
acetone (40 mL), then LiCl (420 mg, 10 mmol) was added
and the mixture stirred and refluxed for 30 min. The sol-
vent was evaporated off in vacuo, and the residue was
partitioned between EtOAc (30 mL and water (10 mL).
The organic phase was separated, dried (MgSO4), filtered,
123

108
J Biomol NMR (2011) 51:105–114
N
N
N
X
Cl
O
O
HO
X
4
5
Br
SSO2CH3
N
O
1
N
O
2
N
O
3
N
O
4,5
Scheme 1 Reagents and conditions: a 1 (1.0 equiv.), Pd(PPh3)4
(0.025 eq.), dioxane, under N2, r.t. 10 min., then 3-Pyridineboronic
acid (1.025 equiv.), aq. 10% Na2CO3 (2.35 equiv.), reflux, 2h, 63%;
b NaBH4 (1.25 equiv.), EtOH, r.t. 15 min. 84%; c MsCl (1.1 equiv.),
Et3N (1.1 equiv.), CH2Cl2, 0°C to r.t. 30 min., acetone, LiCl (2.0
equiv.), reflux 30 min., 45–52%; d NaSSO2CH3 (2.0 equiv.), acetone,
H2O, 40°C, 30–60 min., 36–24%
and evaporated, and the crude residue was further purified
by flash column chromatography (CHCl3/Et2O, 2:1) to
provide radical 4. Yield: 689 mg (52%), pale yellow solid,
mp 87–88°C, Rf : 0.31 (CHCl3/Et2O, 2:1). MS EI m/z (%):
265/267 (M?, 82/28), 250/252(56/21), 235/237 (11/4),
215(100), 184(17). IR (Nujol): 1620, 1560, 1535 (C=C)
cm-1. Composition calculated for C14H18ClN2O: C, 63.27;
H, 6.83; N, 10.54. Found: C, 63.20; H, 6.85; N, 10.52.
For experiments on calmodulin (CaM), a pET21a
(Novagen) vector encoding the 148-residue human (CaM)
protein was created by cloning the gene for CaM from
Xenopus laevis (whose amino acid sequence is identical to
the human protein) into the vector using PCR primers with
BamH1 and NdeI restriction sites. A CaM variant for site-
directed spin labeling, CaM S17C was created with the
QuikChange Site-Directed Mutagenesis Kit (Stratagene).
CaM S17C with uniform 2H/13C/15N labeling was
expressed in BL21-CodonPlus (DE3) RIPL cells (Strata-
gene) at 37°C using standard methods. Cultures of 1 L of
Synthesis of 3-Methanesulfonilthiomethyl -4-(pyridin-3-yl)-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl
Radical [5] (i.e., HO-3606)
2
2
minimal M9 medium using H2O, 15NH4,Cl and H7/13C-
glucose as the sole sources of hydrogen, nitrogen and
carbon, respectively, were induced with 1 mM isopropyl
b-D-1-thiogalactopyranoside (IPTG) at A600nm *0.6, and
harvested by centrifugation 6–9 h later.
To a solution of compound 4 (532 mg, 2.0 mmol) in acetone
(10 mL), NaSSO2CH3 (536 mg, 4.0 mmol) in water (5 mL)
was added and the mixture was kept at 40°C. The reaction
mixture was monitored byTLC. After consumption ofmostof
the starting material (*30 min), the acetone was evaporated
off in vacuo, and the residue was partitioned between CHCl3
(30 mL) and brine (10 mL). The organic phase was separated,
dried (MgSO4), filtered, and evaporated. The resulting residue
wasfurtherpurified byflash column chromatography(hexane/
EtOAc and CHCl3/Et2O)togivethe methanethiosulfonate[5].
Yield: 245 mg (36%), pale yellow solid, mp-125–127°C, Rf
0.12 (CHCl3/Et2O, 2:1). MS EI m/z (%): 341 (M?, 29),
326(10), 346 (39), 215(100), 79(42). IR (Nujol): 1615, 1560,
1535 (C=C) cm-1. Composition calculated for C15H21N2
O3S2: C, 52.76; H, 6.20; N, 8.20; S, 18.78. Found: C, 52.53; H,
6.13; N, 8.05; S, 18.55.
Cells wereresuspended in*25 mL of 40 mM Tris pH7.4,
2 mM EDTA, 2 mM dithiothreitol (DTT), and 19 complete
protease inhibitor (Roche), and lysed using a microfluidizer.
The lysate was cleared by centrifugation and loaded onto a
DEAE ion-exchange column (GE Life Sciences) pre-equili-
brated with 20 mM Tris, pH 7.4, 80 mM (NH4)2SO4 and
1 mM MgCl2. Protein was eluted with a linear 0–100% gra-
dient over 10 column volumes with a high-salt buffer con-
taining 1.2 M (NH4)2SO4. Fractions containing CaM were
pooled and loaded onto phenyl-Sepharose resin (GE Life
Sciences) pre-equilibrated with 50 mM Tris, pH 7.4, 2 mM
CaCl2 and 100 mM NaCl. CaM was eluted with the addition
of 2 mM EGTA. Protein was stored in the elution buffer plus
0.5 mM tris(2-carboxyethyl)phosphine (TCEP) and 19
complete protease inhibitor.
Sample preparation
CaM S17C (*200 lM) was spin labeled by the addition
of a tenfold molar excess of either ‘‘MTSL,’’ HO-3606,
or (1-Acetoxy-2,2,5,5-tetramethyl-d-3-pyrroline-3-methyl)
methanethiosulfonate (Toronto Research Chemicals,
Altenbach et al. 2001), to generate the spin label side
chains R1, R1p, and a diamagnetic analog of R1, respec-
tively. Labeling reactions were allowed to proceed at room
temperature in the dark for *2 h and tested for completion
by liquid chromatography/mass spectrometry. Unreacted
spin-label was removed with a HiPrep 26/10 Desalting
The genetic construct used for expressing single cysteine
mutants of T4 lysozyme (T4L) in the WT* background
(C54T, C97A) has been described previously (Mchaourab
et al. 1996). The mutants were expressed, purified, and
spin labeled with either the common spin label (1-Oxyl-
2,2,5,5-tetramethyl-2,5-dihydropyrrol-3-ylmethyl) methane
thiosulfonate (sometimes referred to as ‘‘MTSL’’ or
‘‘MTSSL’’) (Berliner et al. 1982) or HO-3606 according to
the method described previously (Fleissner et al. 2009).
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J Biomol NMR (2011) 51:105–114
109
Column (GE Life Sciences) and the labeled protein was
2
were recorded at 27°C on a Bruker 600 MHz spectrometer
equipped with a triple resonance z-gradient cryoprobe. Two
exchanged into NMR buffer, comprising 5% H2O/95%
1H2O, 100 mM KCl, 0.19 EDTA-free complete protease
inhibitor (Roche), 25 mM HEPES, pH 6.5, and 8 mM
CaCl2, or into EPR buffer containing the same salts and
buffering agents with 1H2O only. NMR samples were
concentrated to 0.3 mM with Amicon Ultra Centrifugal
Filter Units (3 kDa molecular weight cutoff). EPR exper-
iments were performed at a concentration of 0.1 mM.
Experiments were performed in the presence of 1.2-fold
excess (added prior to centrifugation) of a tightly bind-
ing synthetic peptide (KRRWKKNFIAVSAANRFKKIS
SSGAL; Anaspec) corresponding to the CaM-binding
domain (CBD) of human skeletal muscle myosin-light-
chain kinase (skMLCK).
1
time points (separated by 20 ms) were used for the HN-R2
measurements(Iwaharaetal.2007).Inthiscase,nosignificant
differences in 1HN-R2 were observed for CaM incorporating
the diamagnetic control label and without a label (free-cys-
teine) in NMR buffer containing 5 mM DTT.
PRE data analysis
The position, or ensemble of positions, of the nitroxide spin
label that best fits the observed 1HN-C2 PRE rates was com-
1
puted by comparing predicted and observed HN-C2 PREs
using the coordinates of the structure of Ca2?-loaded, peptide-
bound CaM in complex with smooth muscle MLCK (PDB
1
1CDL) (Meador et al. 1992). HN-C2 PREs were back-cal-
EPR spectroscopy
culatedinXplor-NIH(Schwieters etal. 2006; Schwieters et al.
2003) with either a single conformer or a five-conformer
ensemble for the spin label together with the Solomon-
Bloembergen Model Free (SBMF) representation (Iwahara
et al. 2004b). The label and immediately adjacent side chain
coordinate positions were optimized in torsion angle space by
simulated annealing to minimize the difference between
observed and calculated 1HN-C2 PREs, as previously descri-
bed (Iwahara et al. 2004b) with the addition of a weighting
factor for the PRE force constant equal to the square of the
inverse of the expected error in the observed PRE at each
residue (Xplor-NIH PRE force constant option sigma). The
error in the observed PRE was calculated from the peak
intensities at the two time points in the paramagnetic and
diamagnetic samples (Iwahara et al. 2004b) and a minimum
error of 1.5 s-1 was used to account for small sample-to-
samplevariations.InadditiontothePREpseudo-potential, the
target function includes stereochemical restraints, a quartic
van der Waals repulsion term to prevent atomic overlap
between the spin-label and the protein, and a multidimen-
sional torsion angle database potential of mean force (Clore
and Kuszewski 2002). (Note atomic overlap between spin
labels in the five-conformer ensemble is allowed since the
ensemble represents a distribution of states.) To evaluate the
goodness-of-fit, the appropriate reduced v2:
For EPR experiments, *5 lL samples of T4L (*0.5 mM
protein in 50 mM MOPS pH 6.8, 25 mM NaCl) or CaM
2
(*0.1 mM protein in NMR buffer without H2O) were
loaded into glass capillaries (0.6 mm 9 0.84 mm) sealed
at one end. Where indicated, the sample also included 30%
w/v sucrose to increase the viscosity of the medium, a 1.2
molar excess of the previously described CBD synthetic
peptide from human skMLCK, or both. Spectra were col-
lected at X-band (9 GHz) over 100 G on a Bruker Elexys
580 spectrometer fitted with a high-sensitivity resonator
using 20 mW incident microwave power and 1 G field
modulation amplitude at 100 kHz. Sample temperature was
maintained within a single degree of the reported value
using a Bruker ER4131VT temperature controller. EPR
spectra were simulated using the MOMD model (Budil
et al. 1996), using spatially averaged A and g tensor
principal values corresponding to the nitroxide motion
relative to the protein (the time-independent, effective
Hamiltonian) (Hubbell and McConnell 1971).
NMR spectroscopy
Backbone resonance assignments for peptide bound CaM
were transferred from known assignments and verified by
three-dimensional triple resonance heteronuclear correlation
spectroscopy (Clore and Gronenborn 1994) using standard
HNCO, HNCACB, and CBCA(CO)NH pulse sequences.
Transverse 1HN-C2 PRE rates were obtained from the differ-
!
2
Co2bserved ꢁ C2predicted
X
was computed. The number of degrees of freedom M is
1
v2 ¼
ð1Þ
M
robserved
N
1
ences in the HN-R2 transverse relaxation rates between the
given by:
paramagnetic and diamagnetic samples (Clore and Iwahara
2009) using a 3D HNCO-based pulse scheme similar to a
previously described scheme (Hu et al. 2009) except without
TROSY. Compared to most PRE experiments, this experi-
mentaddsa13C’ dimensiontoresolveoverlapping resonances
present in the 2D 1H-15N correlation spectrum of CaM. Data
M ¼ N ꢁ ð3CÞ
ð2Þ
where N is the number of observed PREs, and C, the
number of spin label conformers in the fitting ensemble,
one or five. C is multiplied by three, for the three transla-
tional degrees of freedom involved in fitting each nitroxide
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J Biomol NMR (2011) 51:105–114
position. Because the effective electron relaxation time ss,
which is equal to the electron spin–lattice relaxation time
30% Sucrose
Buffer
T
1e in the case of 1H-C2 PREs for macromolecules, is much
A
2Azz´
longer than the protein rotational correlation time sr
(Iwahara et al. 2007), the PRE correlation time sc
T4L
D72R1
T4L
D72R1p
×2
[=(s-r 1 ? s-s 1 -1
) ] for the calculation of PRE rates was
assumed to be approximately the same as sr (9.9 ns) for
CaM (Chang et al. 2003).
Complete Xplor-NIH scripts and documentation to
introduce the spin label into a protein of known structure,
find the label position(s) that best-fits the intramolecular
PRE data, and compute v2 are available online at
http://nmr.cit.nih.gov/xplor-nih/doc/current.
T4L
D72R1
T4L
D72R1p
×2
B
Results
CaM
CaM
S17R1
S17R1p
×2
×2
EPR demonstrates restricted internal motion of R1p
relative to R1
All ‘‘free’’ nitroxides have similar isotropic three-line
spectra independent of the substituent at the 3 or 4 posi-
tions of the ring. The spin density at these positions is quite
low, and the presence of the pyridyl ring in R1p has little
effect on the EPR spectrum.
CaM
S17R1
CaM
S17R1p
To compare the internal flexibility of the R1p side chain to
that of R1, the solvent-exposed site D72 of T4L was selected
for introduction of the spin labels. This site has previously
been used as a reference since contributions of backbone
fluctuations to the motion of the nitroxide are minimal
(Columbus et al. 2001). The room temperature EPR spectra of
the T4L D72R1p mutant and the previously published D72R1
mutant (Mchaourab et al. 1996) are presented in Fig. 2a. Both
mutants were studied in buffer (Fig. 2a, red traces), but were
also studied in a 30% sucrose solution (Fig. 2a, black traces),
which reduces the effect of protein rotational diffusion on the
spectrum and allows the motion of the nitroxide relative to the
protein to be examined (Mchaourab et al. 1996). Assuming a
spherical geometry for T4L, an estimate of its rotational cor-
relation time (sR) at room temperature according to the
Stokes–Einstein relation is 6–8 ns in buffer and 18–24 ns in
30% sucrose. Fits to multifrequency EPR spectra of spin
labeled T4L yield sR & 10 ns in buffer and approximately 3
times longer in a viscosity corresponding to 30% sucrose,
consistent withpreviouslyreportedvalues(Zhangetal. 2010).
Regardless of the exact values, rotational diffusion within this
time regime will strongly influence the EPR spectrum as
shown in Fig. 2.
Fig. 2 EPR spectra of R1 and R1p labeled proteins. a EPR spectra of
T4L D72R1 (left) and T4L D72R1p (right) at 298 K. b EPR spectra
of CaM S17R1 (left) and CaM S17R1p (right) at 298 K. Spectra
shown in red are of the mutants in buffer, and those in black are in
buffer containing 30% (w/v) sucrose. Shown below the T4L D72R1p
spectra (gray, dashed trace) are simulated spectra according to a
modified effective Hamiltonian model using values for the magnetic
tensor derived assuming an order parameter of 0.76 and no diffusion
axis tilt (Axx = Ayy = 7.07 G, Azz = 34.9 G, gxx = gyy = 2.00667,
gzz = 2.00267). Best-fit values for sr were 5.4 ns (buffer) and 17 ns
(30% sucrose). All spectra are normalized to the same concentration
of nitroxide and have a magnetic field scan width of 100 G. Vertical
scaling factors (92, left side of spectrum) were applied to select
spectra for display purposes
overall lineshape arisesfroma fastanisotropicinternalmotion
of the nitroxide that can be accurately described by an order
parameter (S) and correlation time (si) on the ns time scale
(Columbus 2001). In the fast motion limit, the time-dependent
Hamiltonian describing the motion can be replaced by an
effective, time-independent, Hamiltonian with the A and g
magnetic interactions tensors averaged over motion within a
cone. In this limit, which has been shown to be a reasonable
approximation for T4L D72R1, the value of 2A0zz is a quan-
titative measure of the order parameter, increasing with
increased order (Columbus et al. 2001).
In solutions of 30% sucrose, the T4L mutants D72R1 and
D72R1p have spectra characterized by well-resolved outer
hyperfine extrema with splitting of 2A0zz (Fig. 2) that corre-
spond to nitroxides with the 2p orbital on nitrogen oriented
along the laboratory magnetic field. For T4L D72R1, the
As is evident in Fig. 2a, the spectra of T4L D72R1p and
T4L D72R1 in sucrose are similar except that 2A0zz is
considerably larger for that of T4L D72R1p (64 G for
123

J Biomol NMR (2011) 51:105–114
111
D72R1p and 53 G for D72R1), indicating a higher order for
the internal motion of R1p. This result is consistent with
expectations from the crystal structure of the 4-phenyl
analog of R1 in which the entire side chain was resolved
(see Discussion). Due to this increase in order, the rota-
tional diffusion of the protein, even in 30% sucrose, will
contribute to the EPR spectrum of T4L D72R1p by
reducing 2A0zz. To deduce the 2Az0 z value corresponding to
purely internal motion of the side chain, spectra of T4L
D72R1p were recorded as a function of sucrose concen-
tration from 15 to 35% wt/wt, and then analyzed (Timofeev
and Tsetlin 1983) to give 2A0zz & 70 G, 5 G less than that
in the rigid limit (Kusnetzow et al. 2006). Within the
context of the effective Hamiltonian model for motion in a
cone (Griffith and Jost 1976), this result gives an order
parameter for the internal motion of R1p of S = 0.76,
compared to 0.43 for R1 (Columbus et al. 2001). If this
simple model has merit, then it should be possible to
reproduce the experimental spectra of T4L D72R1p in both
buffer and 30% sucrose using the effective Hamiltonian
parameters corresponding to S = 0.76 and computing the
effect of protein rotational diffusion according to the sto-
chastic Louisville method that is widely used for simulat-
ing the effect of motion on EPR spectra (Budil et al. 1996).
The dashed traces shown in Fig. 2 are fits to the experi-
mental spectra using the effective Hamiltonian parameters
given in the legend and allowing the isotropic sR of the
protein to vary. The best-fit sR values are 5 and 17 ns for
buffer and 30% sucrose, respectively, and both are within
the expected range, considering the simple nature of the
model and the assumed isotropic diffusion of the protein.
Thus, a first-order working model for the overall motion of
the R1p sidechain is a fast, highly-ordered internal motion
of small amplitude modulated by slow rotational diffusion
of the protein. To explore the generality of the ordered
internal motion of R1p, it was placed at several other
solvent-exposed helical sites in T4L (Fig. 3). For R1p at
helical sites 48, 76 and 131 the spectra are very similar to
that at 72, suggesting that the above results will be general
for R1p in rigid helical structures. The spectrum of R80R1p
has a small 2A0zz, indicating higher nitroxide mobility com-
pared to the others. This is likely due to contributions from
backbone flexibility at this C-terminal site where the crys-
tallographic thermal factors are relatively high. The spectrum
of CaM S17R1 is similar to other non-interacting, solvent
exposed helical sites that exhibit fast (*2 ns), weakly
ordered (S\ 0.5) motion. As for T4L, the spectra of CaM
S17R1p exhibit reduced mobility compared to those of the
analogous R1 mutant (Fig. 2b). Remarkably, the spectra of
CaM S17R1p are similar to those of T4L D72R1p. Because
T4L and CaM are of similar size and the sites in each case
are located at the solvent exposed surface of a helix, the
internal motions relative to the protein must be comparable.
A single label conformation accounts
for the experimental PREs
1
The HN-C2 PRE rates for CaM S17C reacted with the R1
side chain label, CaM S17R1, and with the pyridyl label,
CaM S17R1p, are presented in Fig. 4. In the Ca2?-loaded
and peptide-bound state, CaM adopts a rigid structure in
which the hydrophobic clefts of the N- and C-terminal
domains of CaM close around the target peptide (Ikura
S17C
A
N-terminal domain
C-terminal domain
60
40
20
0
5 conf:
2
χ
red = 1.22
R1
1 conf:
2
χ
red = 1.59
0
20
40
60
80
100
120
140
B
60
40
20
0
5 conf:
2
χ
red = 1.28
R1p
1 conf:
2
χ
red = 1.29
T4L
T4L
K48R1p
R80R1p
0
20
40
60
80
100
120
140
residue number
Fig. 4 Comparison of observed and calculated PRE data for Ca2?
-
loaded, peptide-bound CaM with a spin label conjugated to S17C:
a S17R1; b S17R1p. Experimental PRE profiles are shown as circles
(error bar, 1 SD). PREs too large ([60 s-1) to be accurately
measured are plotted at the top of the charts. PRE profiles back-
calculated from the structures of CaM (Ca2?-loaded, peptide-bound)
are displayed as solid lines using either a single conformer (blue) or 5
conformer ensemble (red) to represent the spin label. Reduced v2
values are given for each of the fits. Note that use of a multiple-
conformer ensemble representation significantly improves the fit to
the experimental data in the case of R1 but not R1p
T4L
R76R1p
T4L
V131R1p
Fig. 3 EPR spectra of T4L R1p mutants in a buffered solution
containing 30% sucrose at 295 K. Solvent-exposed helical sites in
T4L were chosen for incorporation of the R1p spin label. The
magnetic field scan width is 100 G
123

112
J Biomol NMR (2011) 51:105–114
et al. 1992; Meador et al. 1992). Since the labels are
attached in the same S17C position, the pattern of PREs
arising from S17R1 and S17R1p are similar. Quantita-
tively, however, the effect of the larger conformational
dynamics of R1 compared to R1p on the observed PREs
can be easily distinguished.
specific reagent for generating R1p (HO-3606, Scheme 1)
is a 4-pyridyl analog of the commonly used MTSL reagent
that generates the R1 spin label. Although R1 and R1p
differ only by the 4-subsituent, the EPR spectra of R1p at
solvent-exposed sites in T4L and CaM are dramatically
different from those for the analogous R1 mutants, exhib-
iting highly restricted motion relative to the protein
(Fig. 2). This result is consistent with the crystal structure
of the 4-phenyl analog at T4L V131, in which the entire
side chain is resolved in a single rotamer (Fleissner 2007)
(PDB accession code 1ZUR). In contrast, two rotamers
were observed in the crystal structure of R1 at the same site
though the nitroxide rings of neither rotamer was resolved
due to disorder (Fleissner et al. 2009). Interestingly, the
EPR spectra of the rigidly attached R1p mutants are
strikingly similar to mutants bearing the 4-phenyl analog of
R1 (Columbus et al. 2001), suggesting that the conforma-
tional space of the R1p side chain is also restricted. Indeed,
the intramolecular PREs observed for CaM S17R1p are
fully accounted for within experimental error by a single
nitroxide position, and the fit cannot be improved by
introducing an ensemble representation. This is in sharp
contrast to the PREs arising from S17R1 where a multiple
conformer ensemble is required and yields a significantly
better fit than a single conformer representation.
Assuming a single, static conformation of the side chain,
CaM S17R1 shows a reasonable fit with a reduced v2 of
1.59 (Fig. 4, top, blue line) (a value of 1.0 suggesting a
perfect fit within the error of the measurement). Though the
fit is good overall, the observed PREs at positions 67, 68,
and 69 are clearly not well fit, suggesting that the nitroxide
position may sample many different states with distinct
PREs that cannot be captured by a single structure. By
introducing an ensemble of five (equally weighted) con-
formations, the fit to the data for S17R1 can be improved
such that the predicted PREs are within the experimental
error in this region (Fig. 4a, red line). The reduced v2 for
the five-conformer ensemble drops to 1.22, indicating that
the fit is quantitatively superior to the fit using a single
conformation, and hence a multiple conformer ensemble is
necessary to quantitatively treat the PREs for S17R1.
In contrast, the PREs for CaM S17R1p can be quanti-
tatively accounted for by a single conformation of the
label, resulting in a v2 of 1.29 (Fig. 4b, blue line). This
value is considerably smaller than the v2 for a single
conformer representation of S17R1 and comparable to the
v2 for the five-conformer ensemble. Unlike in the case of
S17R1, fitting an ensemble of five conformations of the
label to the observed PREs for S17R1p leaves the fit
essentially unchanged (red and blue lines overlap) and does
not improve the reduced v2, indicating that a single con-
formation of the R1p spin-label side chain is sufficient to
account for the observed PREs.
At any surface helical site, the assumption that R1p can be
treated as a occupying a single position for PRE studies can be
1
quickly determined by combining a pair of HN-R2 experi-
ments to compute intramolecular (or intradomain) PREs with
the Xplor-NIH scripts we have provided (see Methods). Since
quantitative calculation of PREs for a label with a single
nitroxide position does not require the representation of a
conformational ensemble of labels nor a model free analysis
incorporating order-parameters for the nitroxide-proton vec-
tors, we propose that site directed spin-labeling with HO-3606
to form the nitroxide bearing side chain R1p will dramatically
simplify the quantitative procedures needed for analyzing
intra- and intermolecular PRE data.
Discussion
Recent studies have demonstrated that PREs can not only
provide sensitive, long-range structural restraints (Iwahara
et al. 2004b), but can also allow transient, sparsely-popu-
lated structures to be observed (Clore 2011; Clore and
Iwahara 2009). However, a quantitative description of the
PREs arising from most spin labels (e.g., R1) typically
requires the use of an ensemble of spin label conformers to
represent the positions of the nitroxides due to the inherent
conformational flexibility of the spin label side chain. Here
we introduce and characterize a new, rigid spin label for
proteins that, when placed at solvent-exposed helical
positions, can simplify PRE data analysis. In contrast to
other rigidly attached, lanthanide labels developed for PCS,
only a single cysteine is required to incorporate this new,
disulfide-linked spin label, designated R1p; the thiol-
Acknowledgments We thank Vincenzo Venditti and Mengli Cai
for helpful suggestions, Dusty Baber and Jinfa Ying for assistance
´
with NMR spectrometers, and Evan Brooks and Maria Balog for
expert technical assistance. This work was in part supported by funds
from the Intramural Program of the NIH, NIDDK and the Intramural
AIDS Targeted Antiviral Program of the Office of the Director of the
NIH (to G.M.C.), NIH grants R01EY05216 (to W.L.H.), and the Jules
Stein Professorship Endowment (to W.L.H.). The synthesis of new
spin label reagents was supported by Hungarian National Research
Funds (OTKA K81123).
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