Aromatic ring-flipping in supercooled water: Implications for NMR-based structural biology of proteins

We have characterized, for the first time, motional modes of a protein diss olved in supercooled water: the flipping kinetics of phenylalanyl and tyros inyl rings of the 6 kDa protein BPTI have been investigated by NMR at tempe ratures between -3 and -16.5 degreesC. At T = -15 degreesC, the ring-flippi ng rate constants of Tyr 23, Tyr 35, and Phe 45 are smaller than 2 s(-1), i .e., flip-broadening of aromatic NMR lines is reduced beyond detection and averaging of NOEs through ring-flipping is abolished. This allows neat dete ction of distinct NOE sets for the individual aromatic H-1 spins. In contra st, the rings of Phe 4, Tyr 10, Tyr 21, Phe 22, and Phe 33 are flipping rap idly on the chemical shift time scale with rate constants being in the rang e from approximately 10(2) to 10(5) s(-1) even at T = -15 degreesC. Line wi dth measurements in 2D [H-1,H-1]-NOESY showed that flipping of the Phe 4 an d Phe 33 rings is, however, slowed to an extent that the onset of associate d line broadening in the fast exchange limit is registered. The reduced rin g-flipping rate constant of Phe 45 in supercooled water allowed very precis e determination of Eyring activation enthalpy and entropy from cross relaxa tion suppressed 2D [H-1,H-1]-exchange spectroscopy. This yielded DeltaH(dou ble dagger) = 14 +/- 0.5 kcal.mol(-1) and DeltaS(double dagger) = -4 +/- 1 cal.mol(-1).K-1, i.e., values close to those previously derived by Wagner a nd Wuthrich for the temperature range from 4 to 72 degreesC (DeltaH(double dagger) = 16 +/- 1 kcal.mol(-1) and DeltaS(double dagger) = 6 +/- 2 cal.mol (-1.)K(-1)). The preservation of the so far uniquely low value for ASQ indi cates that the distribution of internal motional modes associated with the ring flip of Phe 45 is hardly affected by lowering T well below 0 degreesC. Hence, if a globular protein does not cold denature, aromatic flipping rat es, and thus likely also the rates of other conformational and/or chemical exchange processes occurring in supercooled water, can be expected to be we ll estimated from activation parameters obtained at ambient T. This is of k een interest to predict the impact of supercooling for future studies of bi ological macromolecules, and shows that our approach enables one to conduct NMR-based structural biology at below 0 degreesC in an unperturbed aqueous environment. A search of the BioMagResBank indicated that the overwhelming majority of the Phe and Tyr rings (>95%) are flipping rapidly on the chemi cal shift time scale at ambient T, while our data for BPTI and activation p arameters available for ring-flipping in Iso-2-cytochrome c reveal that in these smaller proteins a total of six out of seventeen rings (similar to 35 %) are "frozen in" at T = -15 degreesC. This suggests that a large fraction of Tyr and Phe rings in globular proteins that are flipping rapidly on the chemical shift time scale at ambient T can be effectively slowed in superc ooled water. The present investigation demonstrates that supercooling of pr otein solutions appears to be an effective means to (i) harvest potential b enefits of stalled ring-flipping for refining NMR solution structures, (ii) recruit additional aromatic rings for investigating protein dynamics, and (iii) use multiple slowly flipping rings to probe cold denaturation. The im plications for NMR-based structural biology in supercooled water are addres sed.