The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold.

This study examines acrylamide quenching of tryptophan room-temperature phosphorescence in proteins and the role that factors such as long-range interactions and environment-dependent quenching efficiency might play in the interpretation of bimolecular quenching rate constants in terms of hindered quencher migration through the globular fold. The distance dependence of the through-space quenching rate is evaluated by studying the effects of acrylamide on the phosphorescence intensity and decay kinetics of the indole analogue 2-(3-indoyl)ethyl phenyl ketone in propylene glycol/buffer glasses, at 120 K. Both steady-state and kinetic data are satisfactorily fitted by an exponential distance dependence of the rate, k(r) = k 0 exp[−(r − r 0)/r e], with a contact rate k 0 = 1.2 × 108 s-1 and an attenuation length r e = 0.29 Å. For a phosphorescence lifetime of 5 s, this rate yields an average interaction distance of 10 Å. The rate is temperature dependent, with k 0, estimated from the bimolecular quenching rate constant (P k q) of Trp analogues in liquids, increasing by about 10-fold from 120 to 293 K. Solvent effects on the quenching efficiency are tested with Trp analogues in water, propylene glycol, and dioxane. The quenching efficiency per collisional encounter is about 0.20 for water, 0.35 for propylene glycol, and drops to 0.025 in the aprotic, least polar dioxane. Acrylamide quenching rate constants are determined for a series of proteins and for experimental conditions appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to P k q = 1.5 × 109 M-1 s-1 for Trp in the solvent, the magnitude of P k q for protected Trp residues in proteins ranges from a maximum of 6 × 104 M-1 s-1, for the most superficial W59 of RNase T1, to 10-1 M-1 s-1 for the most internal W109 of alkaline phosphatase. For most proteins, theoretical estimates of P k q based on the distance dependence of the rate exclude any quenching contribution from through-space interactions by acrylamide in the solvent. This finding, together with a clear correlation between P k q and other indicators of molecular flexibility, implies that in the millisecond-second time scale of phosphorescence acrylamide can migrate through the macromolecule and that its rate is a measure of the frequency and amplitude of the structural fluctuations underlying diffusional jumps. The origin of the discrepancy between fluorescence and phosphorescence quenching rates in proteins is discussed, and an alternative interpretation of fluorescence quenching data is provided.

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Oxygen quenching of protein phosphorescence was determined for the buried Trp residues of apoazurin, liver alcohol dehydrogenase, and alkaline phosphatase as a function of temperature (0-50°C) and applied pressure (up to 3 kbar). Accurate control of the oxygen concentration in solution, by a method that employs an internal protein reference, largely confirms the small bimolecular quenching rate constants (k q ) reported previously for these proteins. Wide variations in flexibility of the globular fold, as attained from protein to protein or by changing external conditions of temperature and pressure, establish that the magnitude of k q is directly correlated to the rigidity of the protein matrix surrounding the chromophore and demonstrates that the quenching rate constant is limited by hindered migration of oxygen through compact regions of the polypeptide. The magnitude of k q implies that O 2 diffusion in proteins can be slowed over 1000-fold relative to water and much more than was inferred from the corresponding fluorescence quenching rate. The activation enthalpy for the structural fluctuations underlying O 2 diffusion in proteins ranges between 9 and 12 kcal mol -1 , similar among the three proteins but larger than the 3 kcal mol -1 for O 2 diffusion in water. The activation volumes, obtained from the pressure dependence of k q , are largest and positive at 50°C and below 2 kbar, but decrease monotonically at higher pressure and at lower temperature. This behavior, together with a similar magnitude of the activation volumes among proteins with different internal mobility are interpreted as to indicate an essential role of internal water molecules in conferring flexibility to protein structure.

O-Acetylserine sulfhydrylase A (OASS-A) is a pyridoxal 5'-phosphate- (PLP-) dependent enzyme that catalyzes the last step in the synthesis of L-cysteine, the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide. The phosphorescence properties of the two tryptophans of wild-type OASS-A, W51 and W162, and of W162 in the W51Y mutant protein have been characterized over the temperature range 170-273 K. In glasses at 170 K, the apoenzyme exhibits a phosphorescence spectrum which is the superposition of two spectra with well-resolved 0,0 vibronic bands centered at 405 and 410 nm, the blue lambda max suggesting that one of the two Trp residues in OASS-A is in a polar pocket, while the other is in a relatively hydrophobic pocket. The presence of PLP in the OASS-A holoenzyme reduces the intrinsic fluorescence by 40-45%, but the spectrum is unaltered except for the appearance of the internal Schiff base ketoenamine fluorescence band centered at 484 nm. The phosphorescence is strongly quenched by PLP, with about 70% reduction in intensity and lifetime. Further, the phosphorescence spectrum of the holoprotein exhibits a single and narrow 0,0 vibronic band centered at 405 nm and a broad band in the 450-550-nm range resulting from delayed fluorescence of the ketoenamine tautomer of the internal Schiff base, sensitized by triplet-singlet energy transfer from tryptophan to the ketoenamine tautomer of PLP. Comparison with data obtained for the W51Y mutant strongly suggests that the 405-nm phosphorescence band derives from W162, and that W51 in the wild type is entirely quenched either by singlet or triplet energy transfer to PLP or by some local group in the protein. From the rate of energy transfer, the separation between W162 and PLP is estimated to be about 25 A. Substrates other than OAS affect only the intensity of the coenzyme fluorescence band (484 nm) and the intensity of delayed fluorescence relative to that of phosphorescence, effects that are attributable to changes in fluorescence quantum yield of the ketoenamine chromophore. Addition of OAS, on the other hand, leads to a splitting of the 0,0 vibronic band in the phosphorescence spectrum of W162, yielding poorly resolved peaks at 406 and 408.5 nm, indicating thereby a change in the environment of the tryptophan residue and therefore in the conformation of the macromolecule as the internal Schiff base is converted to the alpha-aminoacrylate Schiff base. In buffer at 273 K, both the fluorescence and phosphorescence spectra relax to longer wavelengths and the phosphorescence lifetime is reduced to a few milliseconds, all indications that W162 is in a flexible region of the macromolecule, probably in close proximity to the aqueous interface. The phosphorescence lifetime in fluid medium reveals conformational heterogeneity in OASS-A and unveils important structure modulating effects of cofactor, substrates, and pH. Binding of PLP to the apoprotein increases the rigidity of the polypeptide in the region of W162 (in agreement with the greater thermal stability o...