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Vol. 54 No.

2/2007, 235244

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Attaching a spin to a protein site-directed spin labeling in structural

Aleksander Czogalla1, Aldona Pieciul1, Adam Jezierski2 and
Aleksander F. Sikorski1,3
1Faculty of Biotechnology, and 2Faculty of Chemistry, University of Wrocaw, Wrocaw, Poland;
3Academic Centre for Biotechnology of Lipid Aggregates, Wrocaw, Poland

Received: 19 April, 2007; revised: 29 May, 2007; accepted: 11 June, 2007

available on-line: 14 June, 2007

Site-directed spin labeling and electron paramagnetic resonance spectroscopy have recently expe-
rienced an outburst of multiple applications in protein science. Numerous interesting strategies
have been introduced for determining the structure of proteins and its conformational changes at
the level of the backbone fold. Moreover, considerable technical development in the field makes
the technique an attractive approach for the study of structure and dynamics of membrane pro-
teins and large biological complexes at physiological conditions. This review focuses on a brief
description of site-directed spin labeling-derived techniques in the context of their recent appli-

Keywords: site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy, protein structure

Introduction directed spin labeling (SDSL) has emerged as a

powerful method for investigating protein structure
The rapid development of structural meth- and its dynamics, allowing resolution at the level
ods, such as NMR and X-ray crystallography, results of the backbone fold (Hubbell & Altenbach, 1994).
in substantial increase of the speed of new protein The absence of unpaired electrons in most biological
structure determination. However, some issues re- materials would appear to impede the applicability
main generally unattainable to these powerful meth- of EPR methods, but in fact it is advantageous. La-
ods. The fact that the conformation found in the beling of selected molecules on specified sites with
crystallized molecules may not represent the biologi- a small spin label that has a simple EPR signal, to-
cally active one, and difficulties in elucidation of the gether with the limited number of spins contribut-
structure of high molecular mass proteins in solu- ing to the EPR experiment enables a broad range of
tion and of membrane proteins are the major prob- structural problems to be solved. Because the elec-
lems to solve. Moreover, methods that complement tron has a relatively high magnetic moment, EPR
the static protein structures in crystals to offer a dy- provides the necessary sensitivity to yield spectra
namic representation of proteins in solution and in with a good signal-to-noise ratio using less than 1
large, functionally active supramolecular complexes nanomole of a protein.
are currently of exceptional interest. Protein EPR spectroscopy, based mainly on
Over the past decade, electron paramagnetic nitroxide spin labeling, yields information about the
resonance spectroscopy (EPR) combined with site- nitroxide mobility (Columbus & Hubbell, 2002), its

Corresponding author: A.F. Sikorski, Faculty of Biotechnology, University of Wrocaw, S.F. Przybyszewskiego 63/77, 51-
148 Wrocaw, Poland; phone: (48 71) 375 6233; fax: (48 71) 375 6208; e-mail:
Abbreviations: CW EPR, continuous wave electron paramagnetic resonance; DEER, double electron-electron resonance;
DQC, double quantum coherence; DSDSL, double site-directed spin labeling; EM, electron microscopy; EPR, electron
paramagnetic resonance; MOMD, microscopic order macroscopic disorder; MTSL, 3-methylthiosulfonyl-1-oxyl-2,2,5,5-te-
tramethyl-3-pyrroline; NMR, nuclear magnetic resonance; SDSL, site-directed spin labeling; TOAC, 2,2,6,6-tetramethylpi-
peridine-1-oxyl-4-amino-4-carboxylic acid.
A. Czogalla and others
236 2007

solvent accessibility (Oh et al., 2000; Malmberg &

Falke, 2005), the polarity of its microenvironment,
and the distance between two nitroxides in double
site-directed spin labeling (DSDSL) (Steinhoff, 2004).
Thus, obtaining a set of EPR data of spin labeled
variants of the examined protein is sufficient to de-
termine its secondary structure elements, relative
orientations of the latter and the overall topography
of the protein. These methods are appropriate for
studying even large membrane proteins or complex-
es with a great sensitivity to dynamic fluctuations of
its structure. This paper reviews briefly the promis-
ing EPR methods for structural biology of proteins
in the context of the major representative applica-

Spin labeling

Initially, biological EPR measurements were

limited to metalloproteins possessing its own para- Figure 1. Nitroxide-based spin labels commonly used in
SDSL experiments.
magnetic centres or to proteins with naturally oc-
A. proxyl-iodoacetamide (IAP); B. proxyl-maleimide
curing cysteine residues required for capturing spin (MSL); C. proxyl-methanethiosulfonate (MTSL). D. The
labels. Since the application of molecular biology structure of the R1 side chain produced by the reaction
(which allowed engineering of cysteine residues at of MTSL with the sulfhydryl group of a cysteine residue.
targeted sites in proteins) combined with EPR spec- The dihedral angles associated with each bond are defined
as 15.
troscopy, SDSL has become a powerful new tool
in structural biology (Altenbach et al., 1990). The
cysteine scanning method, where selected residues
within the amino-acid sequence are substituted with phase peptide synthesis, alternatively together with
cysteine residues, is easy to use and therefore quite recombinant synthesis of proteins (Becker et al., 2005)
common. Cysteine residues in the protein of inter- or by using synthesized amino acylated tRNA for
est can be subsequently modified with a variety of spin-labeled protein expression in living cells (Shafer
sulfhydryl-specific nitroxide spin labels (see Fig. 1). et al., 2004). By those means, individually designed
However, the methanethiosulfonate spin probe spin labeled side chains can be incorporated into
(MTSL) (Berliner et al., 1982) is currently most often specific sites of a protein. The reduced residual mo-
utilized. The resulting side chain, designated as R1, tion and the defined orientation of such side chains
is comparable to phenylalanine or tryptophan side may improve measurements of inter-spin distances
chains in molecular volume and exerts a negligi- and orientations of the nitroxides relative to each
ble influence on the biological activity and stability other or with respect to the protein backbone.
as well as does not change the secondary structure
(Mchaourab et al., 1996; Bolin et al., 1998). Moreover,
the properties of R1 related to its enhanced sensi- Mobility
tivity to protein backbone motion and secondary
structure facilitate determination of subtle structural The EPR line shape of the nitroxide side
details from the shape of EPR spectra (Owenius et chain is sensitive to the local environment. The
al., 1999; Langen et al., 2000). As indicated in Fig. 1, label mobility appears to be determined by both
there is a rotational freedom of the 4 and 5 tor- backbone motions and steric restraints of neighbor-
sion angles in the R1 side chain, but the interaction ing residues, depending on the length and flexibil-
between S and the C proton limits the 1, 2 and ity of the linker between the nitroxide and the pro-
3 rotamers. tein backbone (Berliner & Reuben, 1989; Hubbell et
Most recently, an interesting alternative for al., 1996). The correlation between the side chain
cysteine spin labeling has been developed. This is mobility, including effects due to the motional rate,
based on incorporation of unnatural amino acids amplitude and geometry, and the protein structure
into proteins. Tailor-made amino acids carrying a has been explored in most details in T4 lysozyme
spin label, such as TOAC (McNulty & Millhauser, (Mchaourab et al., 1996; 1999). Spin labels on the
2000), can be incorporated into the protein by solid- protein surface or in loops between secondary
Applications of site-directed spin labeling in structural biology
Vol. 54 237

structure elements display a high degree of mo- (Rink et al., 1997; Columbus & Hubbel, 2004; Inan-
bility, which results in a small apparent hyperfine ami et al., 2005) and structures (Jiang et al., 1997;
splitting and line width of the EPR spectrum. On Jayasinghe & Langen, 2004) as well as in the fold-
the other hand, a strong interaction of the nitroxide ing processes (Morin et al., 2006).
with neighboring side chains or backbone atoms as A more quantitative understanding of the
found in the protein interior or at subunit interfac- motions that generate a given line shape needs a
es is indicated by a broadened line and increased simulation of EPR spectra. Simulations based on
apparent hyperfine splitting (see Fig. 2). Despite dynamic models are in excellent agreement with
the complicated nature of the nitroxide dynamics, the corresponding experimental spectra of spin la-
reflected by the anisotropy of its motion and dis- beled proteins or other biological molecules help-
tribution of the motional states, EPR line shapes ing to get a more precise view of the dynamics of
have been successfully analysed in terms of em- the analysed system (Barnes et al., 1999; Borbat et
pirical parameters. The first of them is the inverse al., 2001). Moreover, molecular dynamics EPR spec-
line width of the central resonance line (H01) (see tra simulations facilitate the study of the influence
Fig. 2). Plotting of this parameter as a function of of the structure adjacent to the spin labeled site on
residue number may reveal the secondary struc- the spectral line shape (Steinhoff et al., 2000; Beier
ture, as the periodicity of the spin label mobility re- & Steinhoff, 2006). Complications of samples may
fers to a sequence of surface, contact or buried sites be described as microscopically ordered but macro-
(Mchaourab et al., 1996; Pfeiffer et al., 1999; Meh- scopically disordered (MOMD). The MOMD model
boob et al., 2005). The separation between the outer has been developed to describe local side chain mo-
hyperfine extrema (2Apar) may also be taken as a tion by an order parameter and an effective correla-
measure of label mobility (see Fig. 2). A measure tion time which are related to the amplitude of mo-
of nitroxide side chain mobility, which emphasizes tion and the rate of movement, respectively (Budil
contributions from the outer hyperfine extrema in et al., 1996). Employing the model to spectra simu-
the spectrum is the inverse second moment (<H2> lations it is possible to determine whether the line
1) (see Fig. 2). This parameter provides a measure shape corresponds to isotropic or anisotropic motion
of the overall breadth of the spectrum (Mchaourab and to detect multiple components of the spectra.
et al., 1996). The reciprocal plot of <H2>1 versus The existence of the latter may refer either to sev-
H01 has been found to group side chains from eral conformational states of the attached spin label
different topographical regions of the protein fold or to the heterogenity of protein conformations. Sev-
into defined sections on the plot demonstrating the eral experimental approaches have been applied to
correlation between these spectral parameters and unambiguously assign the components of the EPR
regions of secondary structure and tertiary contact spectrum to their respective molecular background,
(Aihara et al., 2006; Hubbell et al., 1996; Isas et al., including experiments with spin labels of various
2002) (see Fig. 2). Mobility is not only a sensor structure (Columbus et al., 2001) and studies as a
of conformation, but may also serve as a sensitive function of temperature (Hanson et al., 2006a). Mu-
monitor of conformational changes in both helices tating amino acids adjacent to the spin label helps to

Figure 2. Spin label mobility, as revealed by EPR spectra line shape.

Examples of X-band EPR spectra of (A) motionally restricted spin label of a buried side chain of a helix and (B) in-
creased mobility of an exposed side chain of the same helix forming spectrin lipid-binding domain (Czogalla, unpub-
lished). The separation between the outer hyperfine extrema (2Apar) and the peak-to-peak separation of the central line
width (H0) provide a measure of label mobility. C. Mobility map constructed as a plot of the inverse second moment
of the EPR spectrum (<H2>1) versus inverse central line width (H01), which indicates the correlation between the mea-
sured parameters and regions of protein topology (Isas et al., 2002).
A. Czogalla and others
238 2007

understand the effect of the local environment (Liet- tive for spin label locations deep in the membrane.
zow et al., 2004). The extension of continuous wave Most recently, a ruler for determining the membrane
(CW) EPR to multiple fields or frequencies helped depth and orientation of proteins considering the ef-
to increase the signal-to-noise ratio and to improve fect of oxygen alone has been developed (Nielsen et
spectral resolution. High field studies reveal previ- al., 2005).
ously unattainable details of spin label motion in its The accessibility of a spin labeled side chain
local environment (Mobius et al., 2005; Budil et al., can also be determined via pulsed EPR methods in
2006). W-band EPR measurements appeared to be an approach called saturation recovery (Nielsen et
particularly useful for the detection of anisotropy al., 2004). One of the major advantages of this tech-
of nitroxide motion when bimodal dynamics of the nique is a more precise determination of the side
spin label exists (White et al., 2007). chain conformation and conformational exchange
(Pyka et al., 2005). Another innovative approach is
the multi quantum EPR (Klug et al., 2005), which
Accesibility allows a better resolution of multiple component
spectra and enhanced spectral sensitivity to both T1
The analysis of the collision frequency of a ni- and T2 (spin-lattice and spin-spin relaxation, respec-
troxide side chain with freely diffusing paramagnetic tively).
reagents provides additional structural information. The depth parameter may become insensi-
Expressed in terms of the accessibility parameter () tive when applied to membrane-adsorbed periph-
(Klug et al., 1997), it provides information on protein eral proteins, as the latter are located at the mem-
structure, the tilt of membrane proteins, orientation brane-water interface more than 5 away from the
and position of proteins at the membrane interface lipid phosphates. While the electrostatic potential
(Hubbell et al., 1998). The accesibility of a particu- of a biological membrane surface declines with the
lar spin-labeled site is determined from the increase distance from that surface, the gradient of electro-
of the label T1 relaxation rate, which is proportional static potential is distributed across the extent of the
to the bimolecular collisional exchange with a sec- protein molecule. Thus, the use of charged metal-
ondary paramagnetic reagent and is measured by ion complexes (Lin et al., 1998; Ball et al., 1999) or
changes of the amplitude of the EPR spectrum paramagnetic lipid Ni(II) chelates (Gross & Hubbell,
with increasing microwave power in the presence 2002) appeared to be effective for exploring the ori-
or absence of a relaxing agent. Thus, the relative entation of proteins or protein domains with respect
accessibility of the spin label to the paramagnetic to the membrane surface. EPR accessibility experi-
probe is expressed by the equation: (probe)=P1/2 ments, used collectively with high-resolution protein
(probe)P1/2 (N2), where P1/2 (probe) and P1/2 (N2) structures and molecular dynamics simulations has
are the half-saturating powers for samples contain- emerged as an adequate approach for analyzing in-
ing probe or purged with dinitrogen to remove teractions between proteins and lipid bilayers (Jaud
the paramagnetic species, respectively. Such an ap- et al., 2007).
proach is the most widely utilized one and is known
as the power saturation method (Altenbach et al.,
1994; Oh et al., 1996; Qin & Cafiso, 1996; Ball et al., Spin-spin distance
1999). As a rule, the EPR signal amplitude increases
linearly with the square root of incident power, until Distance measurement between two site-di-
it begins to saturate and decreases in intensity. The rected probes in a protein is a powerful method to
microwave power at which the signal amplitude is determine its structure and dynamics. EPR tech-
half-saturated is proportional to the spin-lattice re- niques for estimating distances between spins are
laxation rate of the label. During exposition of the based on the dipolar interactions between them in
spin labeled side chain to paramagnetic reagents, an double site-directed spin labeling (DSDSL) experi-
increase in the saturating power can be observed. ments (Eaton & Eaton, 2000; 2004). Although ex-
Apolar oxygen and highly polar metal chelates, such change interactions may also contribute to the inter-
as Ni(II)EDDA or Cr(C2O4)3, in a water/phospholi- action between two spin labels, they are negligible
pid bilayer system are partitioned between the aque- for relatively long interspin distances without a short
ous and hydrophobic phase according to their prop- through-bond pathway. The electron-electron dipo-
erties. Measurements using one or more reagents, lar interaction results in considerable broadening
with concentration gradients in opposite directions or dipolar splitting of the CW EPR spectrum when
across the biological membranes, may be employed compared with the noninteracting spins. When two
to determine the depth parameter (Malmberg & Fal- nitroxides adopt a specific, rigid geometry, both the
ke, 2005). The latter, which is reflected by the equa- relative distance and orientation between the two
tion = ln[ (O2)/(metal chelate)], becomes posi- spins can be determined (Hustedt & Beth, 1999). The
Applications of site-directed spin labeling in structural biology
Vol. 54 239

recently developed tether-in-a-cone model allows butions, half-field transitions have been successfully
one to analyse EPR spectra in terms of both distance applied (Eaton et al., 1983; Persson et al., 2001). How-
distribution and restricted distributions of the rela- ever, the existence of a conformational heterogen-
tive orientation between probes (Hustedt et al., 2006). ity may shift the average distance values strongly
In other cases, distance determination is followed by toward shorter ones, when the half-field transition
the use of different sets of assumptions. method is applied.
In a frozen state, broadening of a CW EPR Most DSDSL experiments fall into the case
spectrum due to nitroxidenitroxide interactions when spins adopt a statistical distribution of dis-
changes the relative intensities of the three compo- tances and relative orientations. The deconvolution
nents of the spectrum. Considering that the dipo- methods assume that there is sufficient flexibility
lar spin-spin splitting is not resolved and that the in the linkage between the two nitroxides, which is
relaxation rate (T1) is slow relative to the splitting, indeed true for the R1 side chain, and thus the in-
the ratio of peak heights (d1/d) (see Fig. 3B) can be terspin vector orientation is randomly distributed
used to estimate the distance between the two spins relative to the magnetic axes of the paramagnetic
(Kokorin et al., 1972; Sun et al., 1999). centres (Steinhoff, 2004). In the crystal structures
In the cases of relatively short inter-spin dis- of spin-labeled T4 lysozyme, the electron density
tances (up to 8 ), where it is the most important to for the nitroxyl ring was poorly defined (Langen et
separate the dipolar interaction and exchange contri- al., 2000), which is consistent with a distribution of

Figure 3. Distance measurements between two spin labels attached to the helical region of the lipid-binding domain
of spectrin (Czogalla et al., 2007).
EPR spectra of a peptide labeled on the i, i+3 positions measured in 40% sucrose (A) or at liquid nitrogen temperature
(B) with the corresponding spectra of singly labeled peptides (grey). Peak heights d and d1 of the spectrum of the dou-
bly labeled peptide are indicated. C. The algorithm for distance analysis using Fourier deconvolution method (Raben-
stein & Shin, 1995; Xiao & Shin, 2000). Step 1: absorbance spectrum of two interacting labels ((B)) (black) and non-
interacting reference spectrum (S(B)) (grey) are Fourier transformed to *() and S*(), respectively. Dividing *()
by S*() the broadening function in the Fourier space (M*()) is obtained. A Gaussian curve (dotted line) is fit to the
broadening function (M*()) (grey) in order to suppress noise. Step 2: The fit function is reverse-Fourier transformed
to provide the broadening function in real space (M(B)). Step 3: The latter is utilized to estimate average splitting value
(<2B>) and the mean spin-spin distance (<r>) is determined according to the equation, where ge is the isotropic g value
for electrons, is the electron Bohr magneton, and 0 is the permittivity of a vacuum. All EPR spectra are shown with
the y-axis scale adjusted to match peak heights and the broadening of the doubly labeled spectra corresponds to the
average distance between nitroxides about 9 .
A. Czogalla and others
240 2007

conformations. The main idea of the deconvolution peratures at which the spin-spin relaxation time is
methods is to compare the spectra for the doubly long enough to allow echo detection.
spin-labeled proteins with the corresponding singly By using multiple DSDSL techniques a more
labeled samples with noninteracting nitroxides (see complete picture can be obtained, as the CW EPR
Fig. 3). Motional averaging of the dipolar interaction and pulsed EPR methods perfectly complement each
is restricted by freezing the samples (Rabenstein & other and the obtained results are in reasonable
Shin, 1995; Steinhoff et al., 1997) or increasing the agreement (Persson et al., 2001). However, due to
viscosity of solution (Altenbach et al., 2001). An EPR multiple rotameric states of spin labels an uncertain-
spectroscopic ruler method is based on Fourier ity of the backbone-spin distance and difficulties in
deconvolution of the dipolar broadening function the interpretation of the spin-spin distances in terms
for two interacting spins from the dipolar broadened of the protein backbone may occur. Using the mod-
CW EPR spectrum (Rabenstein & Shin, 1995; Xiao & eling strategy to find the lowest energy rotamers, it
Shin, 2000). That function is then used to obtain the is possible to obtain a higher resolution in measure-
mean interspin distance and the variance of its dis- ments of distances and distance distributions (Sale et
tribution. The method has proven to be effective in al., 2005).
the range of 7.525 (Xiao & Shin, 2000; Zhang et
al., 2002) with a standard deviation of 0.9 for the
entire range. An alternative approach consists in fit- Illustrative applications
ting of the simulated dipolar broadened spectrum to
the experimental one by multiplication of the mono- SDSL combined with the described EPR meth-
radical spectrum by a broadening function (Steinhoff ods have been successfully used to determine the
et al., 1997). By that means the average distance as structure of proteins and, more importantly, to fol-
well as the width of the Gaussian distance distribu- low the conformational changes related to their bio-
tion between the attached spin labels is calculated logical activity. The distance measurements between
from the fitted line width function (Radzwill et al., nitroxides has proven to be a valuable approach for
2001). The approaches originally developed for rigid detailed helical geometry analysis (Czogalla et al.,
conditions can be successfully used to estimate di- 2007) as well as for following structural transitions
polar interactions between nitroxides even at physi- in peptides while unfolding (McNulty et al., 2001). A
ological temperatures, provided the rotational cor- combination of line shape, accessibility and distance
relation time of the interspin vector is long enough EPR measurements makes possible a precise location
(Altenbach et al., 2001). Applying the above methods of secondary structure elements and their arrange-
it is necessary to take into account the variable frac- ment into a tertiary fold of flexible proteins such as
tion of incomplete spin-labeled protein, as it may be apolipoprotein A-I (Langerstedt et al., 2007). Using a
a major source of error in most DSDSL experiments. similar set of EPR techniques structural models are
The weaker dipole-dipole interactions at dis- obtained which can be subsequently confronted with
tances longer than 20 result in a gradual loss the corresponding crystallographic results. For exam-
of line broadening. Combination of DSDSL with ple, the crystal structure of the cytoplasmic domain
pulsed EPR techniques increases the measurable dis- of erythrocyte membrane band 3 has been recently
tance range up to 80 . However, such experiments confirmed in solution (Zhou et al., 2005). On the
require specialized EPR spectrometers that only re- other hand, EPR methods appeared to be more re-
cently became comercially available. One of these liable and sufficient to investigate the structure and
methods, double electron-electron resonance (DEER), structural changes of the ABC transporter MsbA in
also called pulsed electron-electron resonance (PEL- bilayers (Dong et al., 2005). Similarly, dynamic mod-
DOR), reveals distance information by producing a els of ligand-induced conformational changes can be
spin echo that is modulated with a periodicity re- obtained (Hurth et al., 2004). SDSL and EPR experi-
lated to the dipolar interaction and the resulting os- ments provide high resolution structural information
cilations in the spin echo amplitude correlate with detailing proteinprotein interactions. This enables
the distance and its distribution (Pannier et al., 2000; identification of molecular arrangements between
Jeschke, 2002). The great sensitivity of the DEER subunits of intermediate filaments at physiological
technique allows studies of membrane proteins in conditions (Hess et al., 2002), interactions between
their native environment (Jeschke et al., 2004). Even arrestin and rhodopsin (Hanson et al., 2006b) and
more sensitive is another pulsed method, double docking between the SecB and SecA chaperones of
quantum coherence (DQC) (Borbart & Freed, 1999; E. coli (Crane et al., 2005).
Chiang et al., 2005) which is especially suitable for Accessibility analysis has provided new ca-
long distance measurements (Hara et al., 2007). The pabilities for positioning and orienting spin-labeled
major limitation of the DEER and DQC methods is proteins or their domains at the membrane-water
the necessity to perform measurements at low tem- interface. Peripheral proteins containing C2 do-
Applications of site-directed spin labeling in structural biology
Vol. 54 241

mains analyzed by this technique include cytosolic Concluding remarks

phospholipase A2 (Malmberg et al., 2003; Nielsen
et al., 2005), protein kinase C (Kohout et al., 2003) Recent technical advances together with
and synaptotagmin I (Rufener et al., 2005; Herrick et widespread applications have established SDSL as
al., 2006). An analogous approach was used to de- a well-developed structural biology technique. EPR
termine membrane binding and penetration of an- measurements can be used not only to obtain the
nexin B12 (Isas et al., 2005; Hedge et al., 2006) and structure of a protein but also to elucidate the mech-
peptides derived from myelin basic protein (Musse anisms of its biological activity, which is often unat-
et al., 2006). tainable from other biophysical techniques. Moreo-
One attractive feature of the SDSL methods ver, SDSL studies can complement other method-
is the ability to follow the changes in the protein ologies (e.g. X-ray crystallography, NMR, cryo-EM)
structure with millisecond timescale. Thanks to yielding a new and powerful class of experimental
time-resolved EPR experiments on doubly labeled approaches. The utility of the method is not limit-
bacteriorhodopsin, it was possible to monitor the ed to proteins, and a great progress has been made
light-induced rotation of its transmembrane heli- in applying SDSL to study nucleic acids and other
ces (Radzwill et al., 2001; Xiao et al., 2000). Simi- biologically active molecules. Taken together, SDSL
larly, structural changes upon proteinprotein emerges as a promising technique for the study of
interaction such as rhodopsintransducer interac- structure and conformational dynamics in a wide
tion (Wegener et al., 2000) and receptor-activated range of biological systems.
conformational cycle of G protein (Oldham et al.,
2006) can be observed. DSDSL studies also indi- Acknowledgements
cated the structural details responsible for the ac-
tivation of rhodopsin and the effect of mutations This study was supported by the State Com-
which cause permanent activation of the visual mittee for Scientific Research (KBN) grant no. 2 P04
transduction cascade (Kim et al., 2004). The struc- 021 27.
ture and function of rhodopsin appeared to be
influenced by detergent-solubilization or lipid re-
constitution, indicating applicability of the EPR References
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