The relation between the three Trp residues in barnase has been characterized by computer studies of the molecular model. Trp-35 is shown to be a lone residue. However, the distance between, and the orientation of Trp-94 and Trp-71, should allow efficient energy transfer in the two directions. The overlap integrals have been calculated from the spectra of the individual Trp residues, by subtracting the spectrum of single Trp mutants from the spectra of the wild-type. A multifrequency phasefluorometric study is performed for wild-type barnase and mutant proteins. The lifetimes of the three tryptophans in the wild-type protein have been resolved. To Trp-35 a single fluorescence lifetime is attributed which varies in the different proteins between 4.3 and 4.8 ns and is pH independent between pH 5.8 and 8.9. Trp-71 and Trp-94 are considered to behave as an energy transfer couple with both forward and reverse energy transfer. To the couple two fluorescence lifetimes are attributed: 2.42 (+/- 0.2) ns and 0.74 (+/- 0.1) ns at pH 8.9, and 0.89 (+/- 0.05) ns and 0.65 (+/- 0.05) ns at pH 5.8. In the mutant Trp-94->Phe the lifetime of Trp-71 is 4.73 (+/- 0.008) ns at high and 4.70 (+/- 0.004) at low pH. In the mutant Trp-71->Tyr the lifetime of Trp-94 is 1.57 (+/- 0.03) ns at high and 0.82 (+/- 0.025) at low pH. From these lifetimes, energy transfer efficiencies can be calculated according to Porter. At pH 8.9 a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was found to be 86% for forward and 4% for reverse transfer (all +/- 2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Foerster. The Fluorescence lifetime of Trp-94, as determined in a mutant which lacks Trp-71, is found to be heavily quenched by the neighboring imidazole group of His-18. The results demonstrate the simultaneous forward and reverse energy transfer between two tryptophan residues and the quenching effect of a neighbor imidazole group.
Protein tau helps to maintain integrity and spacing of microtubules (MT), allowing axonal transport that is essential for neuronal functions. Binding of tau to MT is regulated by dynamic phosphorylation, but the important parameters in normal brain remain elusive. Neurodegenerative tauopathies are diverse in origin, triggered by amyloid in Alzheimer's disease (AD) and by mutations or overexpression of tau–4R in frontotemporal dementia (FTD). Neuronal tau–filaments isolated from brains of patients invariable contain hyper–phosphorylated tau, indicating that deteriorated phosphorylation is a prime event in all tauopathies. We develop yeast as less complex models to study the sequence of events leading from phosphorylation to aggregation of human tau. In our humanized yeast strains, tau is pathologically phosphorylated by endogenous kinases on epitopes typical for tauopathies. Both the phosphorylation status at specific residues and the physical aggregation state of tau is controlled by kinases Mds1 and Pho85, the orthologues of mammalian GSK–3β and cdk5. Paradoxically, deficiency of pho85 leads to hyperphosphorylation and dramatic increased aggregation of tau, due to conformation changes. We further engineered yeast strains as sources of recombinant protein tau with specified physiological phosphorylation states, which is impossible in a bacterial system. Isolated wild–type tau–4R binds to taxol–stabilized MT in a phosphorylation dependent manner. Phosphorylation by mds1 effectively prevents binding, which is restored by dephosphorylation. Tau isolated from pho85 deficient yeast failed to bind MT, corroborating its characteristics inside the cells. Surprisingly, isolated mutant tau–P301L bound avidly to MT in a phosphorylation–independent, non–saturable fashion. Imaging by AFM demonstrated, however, that tau–P301L actually aggregated on the MT and thereby not only increased its apparent binding but moreover led to lateral association and distortion of MT. The data obtained in the yeast model link in vivo and in vitro characteristics of phosphorylation, conformation, aggregation and loss of binding to MT of human protein tau.
Human cyclophilin A (hCypA) contains one tryptophan residue at position 121 (Trp121). The fluorescence intensity of this single tryptophan residue doubles upon binding the clinically important immunosuppressant cyclosporin A (CsA). Trp121 is in close contact to the bound CsA and is well-conserved in almost all immunophilins. The enhancement of the fluorescence intensity upon binding CsA is investigated by steady-state and time-resolved fluorescence measurements. The crystal structures of hCypA and the complex hCypA-CsA are compared. Only Glu120 is strongly influenced by the binding of CsA. The distance between the indole ring and the carboxylate group doubles during complexation. The influence of Glu120 on the fluorescence properties of Trp121 was investigated by pH-titration, and by substituting glutamate into an aspartate and an alanine residue. The fluorescence measurements on the glutamate mutants reveal that the carboxylate group influences the fluorescence properties of Trp121 to a limited extent. The major effect of CsA binding, however, consists in a reshuffling of the populations of microconformations of Trp121 leading to a selective increase of the 1.5 ns lifetime component. This selection is also accompanied by a decreased polarity of the environment and an increase in the radiative rate constant.
