Toward Structural Biology in Supercooled Water

Internal mobility limits the accuracy of NMR1 structures:2 NOEs are quenched, and conformational and/or chemical exchange broaden resonances, thus impeding extraction of conformational constraints. A shift of temperature, T, may move such processes into regimes of very fast or slow exchange on the chemical shift time scale. While a large increase of T is limited by macromolecular stability and excitation of yet additional motions, a decrease well below 0 °C is attainable in supercooled water.3 This promises more accurate NMR structures, a means to freeze out conformations and novel insights into biomolecular dynamics, hydration, and cold denaturation. NMR of small carbohydrates allowed observation of hydroxyl protons,4 but multidimensional spectra of macromolecules have not been reported. Here we show the feasibility of NMR-based structural biology in supercooled water. NMR in supercooled water is hampered by high viscosity, η, yielding long overall rotational correlation times, τc, and line broadening; an exponential, η(T), was fitted to published values3 (Figure 1a). Hydrodynamic theory5 predicts for rigid spherical proteins that τc ) 4π[η(T)]rH/3kT (eq 1). rH is the effective radius with rH )[3VM/(4πNA)] + rw (eq 2), where Vh ) 0.73 cm3/g, M, NA and rw are the protein’s specific volume and molecular weight, Avogadro’s number, and the added radius of a monolayer of water, respectively. With rw ) 3.2 A, eq 2 yields rH ) 17.2 A for 9.4 kDa recombinant ubiquitin.6 To verify that theory applies at 0.5% phage the impact of capillaries6 is reduced. Moreover, * To whom correspondence should be addressed. (1) Abbreviations used: NMR, nuclear magnetic resonance; 1D, 2D, 3D, one-, two-, three-dimensional; HNCA, NMR experiment correlating polypeptide backbone 1HN, 15N, and 13CR chemical shifts; HSQC, heteronuclear singlequantum correlation; NOE, nuclear Overhauser effect; T1, longitudinal nuclear spin relaxation time; T1F, transverse nuclear spin relaxation time in the rotating frame; TROSY, transverse relaxation-optimized spectroscopy; dGTP, 2′deoxyguanosine-5′-triphosphate; dTTP, 2′-deoxythymidine-5′-triphosphate; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate. (2) (a) Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York, 1986. (b) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Clarendon Press: Oxford, 1987. (c) Wagner, G. J. Biomol. NMR 1993, 3, 375-386. (d) Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G.; Skelton, N. J. Protein NMR Spectroscopy; Academic Press: San Diego, 1996. (3) Angell, C. A. In Water: a ComprehensiVe Treatise; Frank, F., Ed.; Plenum Press: New York, 1982; pp 1-82. (4) Poppe, L.; van Halbeek, H. Nat. Struct. Biol. 1994, 1, 215-216. (5) (a) Cantor, P. R.; Schimmel, C. R. Biophysical Chemistry;. Freeman: New York, 1980. (b) Tirado, M. M.; Garcia de la Torre, J. J Chem. Phys. 1980, 73, 1986-1993. (6) 0.8 mM solutions (50 mM K-PO4, pH ) 5.9) of 13C/15N labeled human ubiquitin comprising a C-terminal Ser-(His)6-segment (Martek, MD) were put in glass capillaries (Wilmad, NJ, No. 1365-1.7) or 5 mm tubes (Wilmad, NJ, No. 528). T ) -7 °C (5 mm tube) and -16 °C (capillary tube) could be reached without freezing. 3D HNCA was recorded with a sample in which the volume between the capillaries inside the 5 mm tube was also filled with protein solution; this allowed reaching -12 °C. Pf1 phage (ASLA, Riga, Latvia) solutions (0.5% and 1%; w/v) were prepared in capillaries (10 mM K-PO4, pH ) 7.0). Residual 15N-1H dipolar couplings were measured with a 1.5 mM solution of 15N-labeled ubiquitin (Martek, MD) in a 5 mm tube containing 1.3% (w/v) phage at elevated ionic strength (10 mM K-PO4, pH ) 6.9, 250 mM NaCl); NMR lines broaden at lower ionic strength.14 For detection of imino proton resonances, 10 mM aqueous solutions of dGTP and dTTP in capillaries were used (pH ) 7.0). (7) Kay, L. E.; Torchia, D. A.; Bax, A. Biochemistry 1989, 28, 8972-8979. (8) Szyperski, T.; Luginbuhl, P.; Otting, G.; Guntert, P.; Wuthrich, K. J. Biomol. NMR 1993, 3, 151-164. (9) NMR spectra were recorded on a VARIAN Inova750 spectrometer. In capillaries: 15N T1 and T1F with 1D schemes8 extended for suppression of cross correlated relaxation2d (T1-delays: 31, 95, 213, 290, 379, 480, 592, 852, 1000 ms; T1F-delays: 8,16,23,31,47,55,62,78,94,125 ms; 7 kHz 15N continuous wave spin-lock; total integrals of 1HN resonances between 8 and 9.5 ppm, which excludes side chain amides, were determined; T ) 25,20,15,10,5,0,5,-10,-15 °C.; total measurement time: 96 h). 2D [13C,1H]-HSQC (t1,max (13C) ) 22 ms, t2,max (1H) ) 71 ms, T ) 25, 0, -8, -15 °C.; 30 h total). 2D [15N,1H]-TROSY (t1,max (15N) ) 49 ms, t2,max (1H) ) 48 ms, T ) 25,15,5,5,-6,-7,-8,-11,-15 °C.; 60 h total). ω1,ω2-JHN-coupled HSQC (t1,max (15N) ) 49 ms, t2,max (1H) ) 48 ms, T ) -15 °C.; 24 h). In capillaries with Void Volume filled:6 3D HNCA (t1,max (13C) ) 6 ms, t2,max (15N) ) 24 ms, t3,max (1H) ) 48 ms; T ) 25, -11 °C.; 28 and 144 h, respectively). Pf1 solution in 5 mm tube: 2D [15N,1H]-HSQC without JNH decoupling along ω1(N) (t1,max (15N) ) 46 ms, t2,max (1H) ) 48 ms, T ) 25, -7 °C.; 6 and 24 h, respectively). (10) (a) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12366-12371. (b) Salzmann, M.; Wider, G.; Pervushin, K.; Senn, H.; Wuthrich, K. J. Am. Chem. Soc. 1999, 121, 844-848. Figure 1. Overall rotational tumbling of globular proteins in supercooled water. The freezing point of water (273 K) is indicated. (A) Viscosity, η, of water as a function of T. The dots represent published values.3 The fitted curve represents the indicated exponential function. (B) Rotational correlation time, τc, of ubiquitin6 versus T. Experimental values9 are represented by dots, and the middle curve (asterisk) was obtained from a fit of eq 1 yielding rH ) 17.2 A. The upper (rH ) 18.2 A) and the lower curve (rH ) 16.6 A) enclose the experimental values shown at higher resolution in the insert. Fits were performed with SigmaPlot 4.0. 3230 J. Am. Chem. Soc. 2000, 122, 3230-3231