High-pressure EPR reveals conformational equilibria
and volumetric properties of spin-labeled proteins
John McCoy and Wayne L. Hubbell1
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095
Contributed by Wayne L. Hubbell, December 6, 2010 (sent for review October 5, 2010)
Identifying equilibrium conformational exchange and characteriz-
ing conformational substates is essential for elucidating mecha-
nisms of function in proteins. Site-directed spin labeling has pre-
by some event, but verifying conformational exchange at equili-
brium is more challenging. Conformational exchange (microse-
cond–millisecond) is slow on the EPR time scale, and this proves
to be an advantage in directly revealing the presence of multiple
substates as distinguishable components in the EPR spectrum,
allowing the direct determination of equilibrium constants and
free energy differences. However, rotameric exchange of the spin
label sidechain can alsogive rise to multiple components in the EPR
spectrum. Using spin-labeled mutants of T4 lysozyme, it is shown
that high-pressure EPR can be used to: (i) demonstrate equilibrium
between spectrally resolved states, (ii) aid in distinguishing confor-
mational from rotameric exchange as the origin of the resolved
states, and (iii) determine the relative partial molar volume (Δ¯Vo)
and isothermal compressibility (Δ¯βT) of conformational substates
in two-component equilibria from the pressure dependence of the
equilibrium constant. These volumetric properties provide insight
into the structure of the substates. Finally, the pressure depen-
dence of internal side-chain motion is interpreted in terms of
volume fluctuations on the nanosecond time scale, the magnitude
of which may reflect local backbone flexibility.
tuations on the picosecond–nanosecond time scale and slower
conformational fluctuations on the microsecond and longer time
scale (1–3). Molecular flexibility on these time scales plays a
central role in protein function (4). For example, in recognition-
binding sequences, dynamic disorder on the nanosecond–micro-
second time scale may increase the rate of protein–protein
interactions via a “fly casting” mechanism (5). An emerging
disorder-to-order paradigm for interaction (6) can also give rise
to promiscuity in binding that increases the size of the “inter-
Regulation of protein function is often linked to a conforma-
tional switch triggered by an interaction with a chemical or phy-
sical signal. One mechanistic interpretation of this event is
provided by a “preequilibrium” model, which posits that all pos-
sible conformations of a protein exist at equilibrium with popula-
tions proportional to their relative energies (7). The exchange
(“hopping”) event between different conformers is characterized
by lifetimes in the microsecond–millisecond range (1, 2, 8). In this
model, a conformational switch is viewed as a shift in the relative
populations of existing conformational states rather than the
creation of a new state.
To evaluate the above models and elucidate molecular me-
chanisms of protein function, it is essential to have experimental
means for identifying dynamically disordered sequences and for
characterizing conformational equilibria on a broad range of time
scales. Solution NMR spectroscopy is well-established for this
purpose (9, 10), but it is challenged for many systems of current
interest, including intrinsic membrane proteins in their native
lipid environment, and nonequilibrium systems that evolve in
roteins undergo structural fluctuations that span a wide range
of time scales. Among these motions are fast backbone fluc-
time. For such cases, site-directed spin labeling (SDSL) offers
a promising experimental strategy (11–14).
In the usual implementation, SDSL employs the nitroxide side
chain designated R1 (Fig. 1A). The EPR spectra of R1 in a pro-
tein directly reflect nitroxide motion on the picosecond–nanose-
cond time scale, which overlaps the time domain of fast backbone
fluctuations. Hence, R1 is a direct observer of such motions
and has been used to map sequence-specific backbone motion
in soluble (15) and membrane-bound proteins (14, 16).
An important consequence of the EPR time scale is that
although fast backbone motions are directly reflected in the EPR
spectra, conformational exchange on the microsecond–millise-
cond and longer time scales is too slow to produce relaxation
effects that are reflected in the lineshape; at X-band, exchange
between species with lifetimes >100 ns is in the slow exchange
limit. Instead, the presence of two conformations in equilibrium
will, for particular locations of R1, give rise to two components in
the EPR spectrum, each corresponding to one of the conforma-
tions (17, 18) and of intensity proportional to the population,
permitting the direct determination of the equilibrium constant.
However, two-component EPR spectra can also arise from
equilibrium between two rotameric states of R1 that place the
nitroxide in distinct environments (19, 20). In this report, high-
pressure SDSL-EPR is introduced as a means for distinguishing
conformational and rotameric exchange as the origin of two-com-
ponent EPR spectra and for providing quantitative volumetric
information on conformational substates in equilibrium.
For equilibrium between two states of a system, the pressure-
dependent equilibrium constant KðPÞ relative to that at atmo-
spheric pressure (1 bar) is given to second order in pressure by
where P is the gauge pressure; KðPÞ and Kð0Þ are the equilibrium
constantsatpressuresPandP ¼ 0,respectively;andΔ¯VoandΔ¯βT
are the differences in partial molar volume and partial molar
isothermal compressibility of the two states, respectively, at the
reference pressure and temperature (P ¼ 1 bar and T ¼ 294 K).
According to Eq. 1, application of pressure will produce a rever-
sible shift in the relative populations of states, and this provides
an important test for true equilibrium between states detected in
an EPR spectrum.
Author contributions: J.M. and W.L.H. designed research; J.M. performed research; J.M.
and W.L.H. contributed new reagents/analytic tools; J.M. and W.L.H. analyzed data;
and J.M. and W.L.H. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
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1. Mittermaier AK, Kay LE (2009) Observing biological dynamics at atomic resolution
using NMR. Trends Biochem Sci 34:601–611.
2. Palmer AG, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods
for quantifying microsecond-to-millisecond motions in biological macromolecules.
Methods Enzymol 339:204–238.
3. Bruschweiler R (2003) New approaches to the dynamic interpretation and prediction
of NMR relaxation data from proteins. Curr Opin Struct Biol 13:175–183.
4. Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature
5. Trizac E, Levy Y, Wolynes PG (2010) Capillary theory for the fly-casting mechanism.
Proc Natl Acad Sci USA 107:2746–2750.
6. Wright PE, Dyson HJ (2009) Linking folding and binding. Curr Opin Struct Biol
7. Ma B, Shatsky M, Wolfson HJ, Nussinov R (2002) Multiple diverse ligands binding at a
single protein site: A matter of pre-existing populations. Protein Sci 11:184–197.
8. Lange OF, et al. (2008) Recognition dynamics up to microseconds revealed from an
RDC-derived ubiquitin ensemble in solution. Science 320:1471–1475.
9. Mittermaier A, Kay LE (2006) New tools providenew insights in NMR studies of protein
dynamics. Science 312:224–228.
10. PalmerAG, 3rd,Massi F (2006) Characterization of the dynamics ofbiomacromolecules
using rotating-frame spin relaxation NMR spectroscopy. Chem Rev 106:1700–1719.
11. Fanucci GE, Cafiso DS (2006) Recent advances and applications of site-directed spin
labeling. Curr Opin Struct Biol 16:644–653.
12. Hubbell WL, Cafiso DS, Altenbach C (2000) Identifying conformational changes with
site-directed spin labeling. Nat Struct Biol 7:735–739.
13. Hubbell WL, Gross A, Langen R, Lietzow MA (1998) Recent advances in site-directed
spin labeling of proteins. Curr Opin Struct Biol 8:649–656.
14. Columbus L, Hubbell WL (2002) A new spin on protein dynamics. Trends Biochem Sci
15. Columbus L, Hubbell WL (2004) Mapping backbone dynamics in solution with site-
directed spin labeling. Biochemistry 43:7273–7287.
16. Hubbell WL, Altenbach C, Hubbell CM, Khorana HG (2003) Rhodopsin structure,
dynamics, and activation: A perspective from crystallography, site-directed spin label-
ing, sulfhydryl reactivity, and disulfide cross-linking. Adv Protein Chem 63:243–290.
17. Bridges MD, Hideg K, Hubbell WL (2010) Resolving conformational and rotameric
exchange in spin-labeled proteins using saturation recovery EPR. Appl Magn Reson
18. Lopez CJ, Fleissner MR, Guo Z, Kusnetzow AK, Hubbell WL (2009) Osmolyte
perturbation reveals conformational equilibria in spin-labeled proteins. Protein Sci
19. Guo ZF, Cascio D, Hideg K, Hubbell WL (2008) Structural determinants of nitroxide
motion in spin-labeled proteins: Solvent-exposed sites in helix B of T4 lysozyme.
Protein Sci 17:228–239.
20. Guo ZF, Cascio D, Hideg K, Kalai T, Hubbell WL (2007) Structural determinants of
nitroxide motion in spin-labeled proteins: Tertiary contact and solvent-inaccessible
sites in helix G of T4 lysozyme. Protein Sci 16:1069–1086.
21. Li H, Akasaka K (2006) Conformational fluctuations of proteins revealed by variable
pressure NMR. Biochim Biophys Acta 1764:331–345.
22. Akasaka K (2006) Probing conformational fluctuation of proteins by pressure
perturbation. Chem Rev 106:1814–1835.
23. Fleissner MR, Cascio D, Hubbell WL (2009) Structural origin of weakly ordered
nitroxide motion in spin-labeled proteins. Protein Sci 18:893–908.
24. Langen R, Oh KJ, Cascio D, Hubbell WL (2000) Crystal structures of spin labeled
T4 lysozyme mutants: Implications for the interpretation of EPR spectra in terms of
structure. Biochemistry 39:8396–8405.
25. Zhang ZW, et al. (2010) Multifrequency electron spin resonance study of the dynamics
of spin labeled T4 lysozyme. J Phys Chem B 114:5503–5521.
26. Columbus L, Kalai T, Jeko J, Hideg K, Hubbell WL (2001) Molecular motion of spin
labeled side chains in alpha-helices: Analysis by variation of side chain structure.
27. Bett KE, Cappi JB (1965) Effect of pressure on the viscosity of water. Nature
28. Dadali AA, Barashkova II, Lastenko IP, Wasserman AM (1991) Effect of pressure on the
rotational mobility of spin label in polymer. Eur Polym J 27:1097–1100.
29. Dadali AA, Wasserman AM,BuchachenkoAL, Irzhak VI (1981) Effectof pressure on the
rotational mobility of spin probes in polymers. Eur Polym J 17:525–532.
30. Kato H, Feng H, Bai Y (2007) The folding pathway of T4 lysozyme: The high-resolution
structure and folding of a hidden intermediate. J Mol Biol 365:870–880.
31. Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of
“invisible” excited protein states using HSQC and HMQC experiments. J Am Chem
32. Mulder FA, Mittermaier A, Hon B, Dahlquist FW, Kay LE (2001) Slow internal dynamics
in proteins: Application of NMR relaxation dispersion spectroscopy to methyl groups
in a cavity mutant of T4 lysozyme. Nat Struct Biol 8:932–935.
33. Cellitti J, Bernstein R, Marqusee S (2007) Exploring subdomain cooperativity in T4
lysozyme II: Uncovering the C-terminal subdomain as a hidden intermediate in the
kinetic folding pathway. Protein Sci 16:852–862.
34. Cellitti J, et al. (2007) Exploring subdomain cooperativity in T4 lysozyme I: Structural
and energetic studies of a circular permutant and protein fragment. Protein Sci
35. Lu J, Dahlquist FW (1992) Detection and characterization of an early folding inter-
mediate of T4 lysozyme using pulsed hydrogen exchange and two-dimensional
NMR. Biochemistry 31:4749–4756.
36. Dixon MM, Nicholson H, Shewchuk L, Baase WA, Matthews BW (1992) Structure of a
hinge-bending bacteriophage T4 lysozyme mutant, Ile3-Pro. J Mol Biol 227:917–933.
37. Kawahara K, Tanford C (1966) Viscosity and density of aqueous solutions of urea and
guanidine hydrochloride. J Biol Chem 241:3228–3232.
38. Llinas M, Marqusee S (1998) Subdomain interactions as a determinant in the folding
and stability of T4 lysozyme. Protein Sci 7:96–104.
39. Kato H, Vu ND, Feng H, Zhou Z, Bai Y (2007) The folding pathway of T4 lysozyme: An
on-pathway hidden folding intermediate. J Mol Biol 365:881–891.
40. Visser AJ, Li TM, Drickamer HG, Weber G (1977) Volume changes in the formation of
internal complexes of flavinyltryptophan peptides. Biochemistry 16:4883–4886.
41. Li H, Yamada H, Akasaka K (1999) Effect of pressure on the tertiary structure and
dynamics of folded basic pancreatic trypsin inhibitor. Biophys J 77:2801–2812.
42. Royer CA (2002) Revisiting volume changes in pressure-induced protein folding.
Biochim Biophys Acta 1595:201–209.
43. Taulier N, Chalikian TV (2002) Compressibility of protein transitions. Biochim Biophys
44. Gekko K, Araga M, Kamiyama T, Ohmae E, Akasaka K (2009) Pressure dependence of
the apparent specific volume of bovine serum albumin: Insight into the difference
between isothermal and adiabatic compressibilities. Biophys Chem 144:67–71.
45. Prehoda KE, Mooberry ES, Markley JL (1998) Pressure denaturation of proteins:
Evaluation of compressibility effects. Biochemistry 37:5785–5790.
46. Cooper A (1984) Protein fluctuations and the thermodynamic uncertainty principle.
Prog Biophys Mol Biol 44:181–214.
47. Matsumura M, Matthews BW (1989) Control of enzyme activity by an engineered
disulfide bond. Science 243:792–794.
48. Nicholson H, Anderson DE, Daopin S, Matthews BW (1991) Analysis of the interaction
between charged side chains and the alpha-helix dipole using designed thermostable
mutants of phage T4 lysozyme. Biochemistry 30:9816–9828.
49. Mchaourab HS, Lietzow MA, Hideg K, Hubbell WL (1996) Watching proteins move
using site-directed spin labeling. Biochemistry 35:7692–7704.
50. Yamada H, et al. (2001) Pressure-resisting cell for high-pressure, high-resolution nu-
clear magnetic resonance measurements at very high magnetic field. Rev Sci Instrum
51. Pfund DM, Zemanian TS, Linehan JC, Fulton JL, Yonker CR (1994) Fluid structure
in supercritical xenon by nuclear magnetic resonance spectroscopy and small angle
X-ray scattering. J Phys Chem 98:11846–11857.
52. Schneider DJ, Freed JH (1989) Biological Magnetic Resonance, eds LJ Berliner and
J Reuben (Plenum, New York), Vol 8, pp 1–76.
53. Budil DE, Saxena S, Freed JH (1996) Nonlinear-least-squares analysis of slow motional
EPR spectra in one and two dimensions using a modified Levenberg–Marquardt
algorithm. J Magn Reson Ser A 120:155–189.
54. Kusnetzow AK, Altenbach C, Hubbell WL (2006) Conformational states and dynamics
of rhodopsin in micelles and bilayers. Biochemistry 45:5538–5550.
55. DeLano WL (2002) The PyMOL User’s Manual (DeLano Scientific, Palo Alto, CA).
6 of 6
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