Abstract
Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR (CW-EPR) spectra were used to investigate the solvent effect on the librational motion of human haemoglobin spin-labelled on cysteine β93 with the nitroxide derivative of maleimide, 6-MSL. Protein samples fully hydrated in phosphate buffer solution (PBS), in a 60% v/v glycerol/water mixture and in the lyophilized form were measured at cryogenic temperature in the frozen state. The protein librational motion was characterized by the amplitude–correlation time product, 〈α 2〉τ c, deduced from the ED-EPR spectra. The librational amplitude, 〈α 2〉, was determined independently, from the motionally averaged hyperfine splitting in the CW-EPR spectra, and the librational correlation time, τ c, was derived from the combination of the pulsed and conventional EPR data. Rapid librational motion of small amplitude was detected in all samples. In each case, the librational dynamics was restricted up to 180 K, beyond which it increased steeply for the hydrated protein in PBS and in the presence of glycerol. In contrast, in the dehydrated protein, the librational dynamics was hindered and less dependent on temperature up to ~240 K. In all samples, 〈α 2〉 deviated from small values only for T > 200 K, where a rapid increase of 〈α 2〉 was evident for the hydrated samples, whereas limited temperature variation was shown in the lyophilized samples. The librational correlation time was in the sub-nanosecond regime and weakly dependent on temperature. The results evidence that solvent favours protein dynamics.
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References
Angell CA (2008) Insights into phases of liquid water from study of its unusual glass-forming properties. Science 319:582–587
Banham JE, Jeschke G, Timmel CR (2007) Evidence from EPR that nitroxide spin labels attached to human hemoglobin alter their conformation upon freezing. Mol Phys 105:2041–2047
Bartucci R, Guzzi R, Marsh D, Sportelli L (2003) Chain dynamics in the low temperature phase of lipid membranes by Electron Spin Echo spectroscopy. J Magn Reson 162:371–379
Bartucci R, Erilov DA, Guzzi R, Sportelli L, Dzuba SA, Marsh D (2006) Time-resolved electron spin resonance studies of spin-labelled lipids in membranes. Chem Phys Lipids 141:142–157
Bartucci R, Guzzi R, De Zotti M, Toniolo C, Sportelli L, Marsh D (2008) Backbone dynamics of alamethicin bound to lipid membranes: spin-echo EPR of TOAC spin labels. Biophys J 94:2698–2705
Berliner LJ (ed) (1976) Spin labeling. Theory and applications, vol I. Academic Press, New York
Bridges MD, Hideg K, Hubbel WL (2010) Resolving conformational and rotameric exchange in spin-labeled proteins using saturation recovery EPR. Appl Magn Reson 37:363–390
De Simone F, Guzzi R, Sportelli L, Marsh D, Bartucci R (2007) Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin. Biochim Biophys Acta 1768:1541–1549
Doster W (2010) The protein-solvent glass transition. Biochim Biophys Acta 1804:3–14
Dzuba SA (1996) Librational motion of guest spin probe molecules in glassy media. Phys Lett A 213:77–84
Dzuba SA (2000) Libration motion of guest spin probe molecules in organic glasses: CW EPR and electron spin echo study. Spectrochim. Acta A 56:227–234
Dzuba SA, Tsvetkov YD, Maryasov AG (1992) Echo-induced EPR spectra of nitroxides in organic glasses: model of orientational molecular motions near equilibrium position. Chem Phys Lett 188:217–222
Erilov DA, Bartucci R, Guzzi R, Marsh D, Dzuba SA, Sportelli L (2004a) Echo-detected electron paramagnetic resonance spectra of spin-labeled lipids in membrane model systems. J Phys Chem B 108:4501–4507
Erilov DA, Bartucci R, Guzzi R, Marsh D, Dzuba SA, Sportelli L (2004b) Librational motion of spin-labeled lipids in high-cholesterol containing membranes from echo-detected EPR spectra. Biophys J 87:3873–3881
Fenimore PW, Frauenfelder H, McMahon BH, Young RD (2004) Bulk-solvent and hydration-shell fluctuations, similar to α- and β-fluctuations in glasses, control protein motions and functions. Proc Natl Acad Sci USA 101:14408–14413
Fitter J, Lechner RE, Dencher NA (1999) Interaction of hydration water and biological membranes studied by neutron scattering. J Phys Chem B 103:8036–8050
Frauenfelder H, Petsko GA, Tsernoglou D (1979) Temperature-dependent x-ray diffraction as a probe of protein structural dynamics. Nature 280:558–563
Frauenfelder H, Chen G, Berendzen J, Fenimore PW, Jansson H, McMahon BH, Stroe IR, Swenson J, Young RD (2009) A unified model of protein dynamics. Proc Natl Acad Sci USA 106:5129–5134
Gabel F, Bicout D, Lehnert U, Tehei M, Weik M, Zaccai G (2002) Protein dynamics studied by neutron scattering. Q Rev Biophys 35:327–367
Guzzi R, Bartucci R, Sportelli L, Esmann M, Marsh D (2009) Conformational heterogeneity and spin-labelled -SH groups: pulsed EPR of Na, K-ATPase. Biochemistry 48:8343–8354
Isaev NP, Dzuba SA (2008) Fast stochastic librations and slow rotations of spin-labeled stearic acids in a model phospholipids bilayer at cryogenic temperatures. J Phys Chem B 112:13285–13291
Johnson ME (1978) Librational motion of an immobilized spin label: hemoglobin spin labelled by a maleimide derivative. Biochemistry 17:1223–1228
Khodadadi S, Roh JH, Kisliuk A, Mamontov E, Tyagi M, Woodson SA, Brider RM, Sokolov AP (2010a) Dynamics of biological macromolecules: not a simple slaving by hydration water. Biophys J 8:1321–1326
Khodadadi S, Malkovskiy A, Kisliuk A, Sokolov AP (2010b) A broad glass transition in hydrated proteins. Biochim Biophys Acta 1804:15–19
Kirilina EP, Dzuba SA, Maryasov AG, Tsvetkov YD (2001) Librational dynamics of nitroxide molecules in a molecular glass studied by echo-detected EPR. Appl Magn Reson 21:203–221
Klare JP, Steinhoff HJ (2009) Spin labeling EPR. Photosynth Res 102:377–390
Kulik LV, Salnikov ES, Dzuba SA (2005) Nuclear spin relaxation in free radicals as revealed in a stimulated electron spin echo experiment. Appl Magn Reson 28:1–11
Kurad D, Jeschke G, Marsh D (2003) Lipid membrane polarity profiles by high-field EPR. Biophys J 85:1025–1033
Li DX, Liu BL, Liu YS, Chen CL (2008) Predict the glass transition temperature of glycerol–water binary cryoprotectant by molecular dynamic simulation. Cryobiology 56:114–119
Marsh D (1981) Electron spin resonance: spin labels. In: Grell E (ed) Membrane spectroscopy. Spinger, Berlin, pp 51–142
Marsh D (2001) Polarity and permeation profiles in lipid membranes. Proc Natl Acad Sci USA 98:7777–7782
Parak FG, Achterhold K, Schmidt M, Prusakov V, Croci S (2006) Protein dynamics on different timescales. J NonCryst Solids 352:4371–4378
Paschenko SV, Toporov YuV, Dzuba SA, Tsvetkov YuD, Vorobiev AK (1999) Temperature dependence of amplitudes of libration motion of guest spin-probe molecules in organic glasses. J Chem Phys 110:8150–8154
Poluetkov OG, Utschig LM, Dalosto S, Thumauer MC (2003) Probing local dynamics of the photosynthetic bacterial reaction center with a cysteine specific spin label. J Phys Chem B 107:6239–6244
Rasmussen BF, Stock AM, Ringe D, Petsko GA (1992) Crystalline ribonuclease A loses function below the dynamic transition at 220 K. Nature 357:423–424
Roh JH, Brider RM, Damjanovic A, Thirumalai D, Woodson SA, Sokolov AP (2009) Dynamics of tRNA at different levels of hydration. Biophys J 96:2755–2762
Ruggiero J, Sanches R, Tabak M, Nascimento OR (1985) Motional properties of spin labels in proteins: effects of hydration. Can J Chem 64:366–372
Rupley JA, Careri G (1991) Protein hydration and function. Adv Protein Chem 41:37–172
Steinbach PJ, Brooks BR (1996) Hydrated myoglobin’s anharmonic fluctuations are not primarily due to dihedral transitions. Proc Natl Acad Sci USA 93:55–59
Steinhoff HJ, Lieutenant K, Schlitter J (1989) Residual motion of haemoglobin-bound spin labels as a probe for protein dynamics. Z Naturforsch 44c:280–288
Van SP, Birrell GB, Griffith OH (1974) Rapid anisotropic motion of spin labels. Models for motion averaging of the ESR parameters. J Magn Reson 15:444–459
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Scarpelli, F., Bartucci, R., Sportelli, L. et al. Solvent effect on librational dynamics of spin-labelled haemoglobin by ED- and CW-EPR. Eur Biophys J 40, 273–279 (2011). https://doi.org/10.1007/s00249-010-0644-5
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DOI: https://doi.org/10.1007/s00249-010-0644-5