No abstract

This investigation enquires into the factors that are responsible for the wide range of room-temperature Trp phosphorescence lifetimes (tau) in proteins. By exploiting the enhanced sensitivity and time resolution of phosphorescence measurements, experiments were conducted to evaluate the triplet quenching potential of each amino acid side chain. From the magnitude of the Stern-Volmer rate constant it is concluded that, among the amino acids, quenching reactions at 20 degrees C are quite effective with His, Tyr, Trp, cysteine, and cystine, with rate enhancements of 20 and 50 times when the side chains of Tyr and His, respectively, are in the ionized form. The distance dependence of the quenching interaction, estimated from the quenching of internal Trp residues in proteins separated from the amino acid in solution by a protein spacer of various thickness, emphasizes the very short-range nature of the process. The importance of these side chains, and to some extent that of the peptide linkage, as intrinsic quenchers of Trp phosphorescence in proteins was also confirmed with short synthetic peptides prepared appositely with only one type of these residues. Finally, very short (microseconds) phosphorescence lifetimes of Trp residues in proteins were shown to be invariably associated with the presence of Tyr or Cys in the immediate neighborhood of the chromophore. From a survey of the amino acids that are nearest neighbors to Trp in proteins and the corresponding value of tau it was established that, in the absence of His, Tyr, Trp, and Cys, tau is > or = 1 ms and appears to reflect mainly the local fluidity of the protein structure. Otherwise, tau can be much shorter, and for bulky His, Tyr, and Trp side chains it seems to depend dramatically on the mutual chromophore-quencher orientation. In these cases the triplet decay kinetics is shown to be a complex function of temperature, pH, and flexibility of the protein site.

This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

No abstract

The accessibility of acrylamide to buried Trp residues in proteins, as attested by dynamic quenching of their fluorescence emission, is often interpreted in terms of migration of the quencher (Q) through the globular fold. The quencher penetration mechanism, however, has long been debated because, on one hand, solutes the size of acrylamide are not expected to diffuse within the protein matrix on the nanosecond time scale of fluorescence and, on the other hand, alternative reactions pathways where Q remains in the solvent cannot be ruled out. To test the Q penetration hypothesis, we compared the quenching rates of acrylamide analogs of increasing molecular size (acrylonitrile, acrylamide, and bis-acrylamide) on the buried Trp residues of RNaseT1 and parvalbumin. The results show that the largest molecule, bis-acrylamide, is also the most efficient quencher and that in general the quenching rate is not correlated to quencher size, as expected for a penetration mechanism. Whereas these results rule out significant internal Q migration in the times of fluorescence, it is also demonstrated that up to a depth of burial of 3 A, through-space interactions with acrylamide in the solvent satisfactorily account for the small rate constants reported for these proteins. More generally, this analysis emphasizes that reduced dynamic quenching of protein fluorescence by acrylamide rather than reporting on the structural rigidity of the globular fold reflects the distance of closest approach between the internal chromophore and Q in the aqueous phase.