The microtubule-associated protein tau stabilizes microtubules in its physiological role, whereas it forms insoluble aggregates (paired helical filaments) in Alzheimer's disease. Soluble tau is considered a natively unfolded protein whose residual folding and intramolecular interactions are largely undetermined. In this study, we have applied fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) to examine the proximity and flexibility of tau domains and the global folding. FRET pairs spanning the tau molecule were created by inserting tryptophans (donor) and cysteines (labeled with IAEDANS as an acceptor) by site-directed mutagenesis. The observed FRET distances were significantly different from those expected for a random coil. Notably, the C-terminal end of tau folds over into the vicinity of the microtubule-binding repeat domain, the N-terminus remains outside the FRET distance of the repeat domain, yet both ends of the molecule approach one another. The interactions between the domains were obliterated by denaturation in GdnHCl. Paramagnetic spin-labels attached in various domains of tau were analyzed by EPR and exhibited a high mobility throughout. The data indicate that tau retains some global folding even in its "natively unfolded" state, combined with the high flexibility of the chain.

This study aims to investigate the g xx heterogeneity of the g-tensor commonly observed in high-field electron paramagnetic resonance (EPR) spectra of nitroxide spin-labeled sites in proteins. This heterogeneity is addressed in terms of spin-label populations characterized by different polarity and H-bonding properties of the nitroxide micro-environment. The g xx value for each population is determined from the fit of continuous-wave high-field spectra obtained at 95, 275 and 360 GHz with a series of nitroxide spin-labels covalently attached to different sites in both membrane and water-soluble proteins.

Taking advantage of the improved spectral resolution of high-field electron paramagnetic resonance (EPR) at 95 GHz/3.4 Tas compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the polarity profile in a protein-protein interface is obtained. Nitroxide spin label side chains are introduced at positions 88 to 94 in the AS-1 sequence of the membrane adjacent HAMP domain of the transducer protein, NpHtrII, which is reconstituted in complex with sensory rhodopsin, NpSRII from Natronobacterium pharaonis. Position-dependent variations of the values of the nitroxide magnetic tensor components g.~, and A.: suggest that the spin label side chains at positions 88 to 93 of AS-1 are located between a hydrophobic anda hydrophilic microenvironment. The observed periodicity of the polarity properties of the respective spin label microenvironment agrees with an ~t-helical secondary structure of this part of AS-I and validates a recently published molecular model which locates residues 88 and 91 in the interface between helices F and G of NpSRII and AS-1 of NpHtrII close to the cytoplasmic lipid-water interface.

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