Alain Sillen

The Correct Use of “Average” Fluorescence Parameters

When more than one fluorophore is present or when one fluorophore displays a multiple exponential decay “average” fluorescence parameters are derived, which can be combined with “average” lifetimes for further interpretation. However, two kinds of average lifetimes are used in this context: the intensity and the amplitude average lifetime. In this paper the different average parameters are carefully analyzed and their “best” combinations are derived. These average parameters are analyzed in the context of external and internal dynamic and static quenching, Föster energy transfer and the calculation of the radiative rate constant. The use of the amplitude average lifetime for the analysis of multiple fluorophore‐containing systems and the detection of interactions is discussed.

NMR analysis of a tau phosphorylation pattern

The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimers diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.

Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein.

A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type‐specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated protein.

Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules

In Alzheimer disease (AD)‐affected neurons, the Tau protein is found in an aggregated and hyperphosphorylated state. A common hypothesis is that Tau hyperphosphorylation causes its dissociation from the microtubular surface, with consequently a breakdown of the microtubules (MTs) and aggregation of the unbound Tau. We evaluated the effect of Tau phosphorylation on both tubulin assembly and MT binding. We show that the cyclin‐dependent kinase 2/cyclin A3 kinase complex can generate the AT8 and AT180 AD‐specific phospho‐epitopes and use NMR spectroscopy to validate qualitatively and quantitatively the phospho content of our samples. The simultaneous presence of both epitopes disables the tubulin assembly capacity of Tau in conditions whereby Tau is the driving force for the assembly process but does not, however, inhibit MT assembly when the latter is driven by an increased tubulin concentration. When compared to the isolated MT binding repeats (Kd=0.3 µΜ), the phospho‐Tau retains a substantial affinity for preformed MTs (Kd=11 nM), suggesting that the phosphorylated proline‐rich region still participates in the binding event. Our results hence indicate that the sole phosphorylation at the AT8/AT180 epitopes, although leading to a functional defect for Tau, is not sufficient for its dissociation from the MT surface and subsequent aggregation as observed in AD.—Amniai, L., Barbier, P., Sillen, A., Wieruszeski, J.‐M., Peyrot, V., Lippens, G., Landrieu, I. Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules. FASEB J. 23, 1146–1152 (2009)

Equilibrium and kinetic study of the conformational transition toward the active state of p21Ha-ras, induced by the binding of BeF3- to the GDP-bound state, in the absence of GTPase-activating proteins.

Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595–15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115–117). In this work, we have used a fluorescent active mutant (Y32W) of p21Ha- ras to demonstrate that BeF3 − binds to the GDP·p21Ha-ras complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (K d at 25 °C = 8.1 ± 0.3 mm) and is endothermic (ΔH = 22.3 ± 1.6 kJ mol−1). The similarity between the GTP-bound form and the GDP·BeF3 −-bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP·BeF3 −·p21Ha- ras complex to its final state. Both steps are endothermic (ΔH 1 = 15 ± 2 kJ mol−1 and ΔH 2 = 8 ± 2 kJ mol−1). The kinetically determined enthalpy change of 23 ± 4 kJ mol−1 is in excellent agreement with the equilibrium analysis.

Regions of Tau Implicated in the Paired Helical Fragment Core as Defined by NMR

We have studied the mature Alzheimer‐like fibers of tau by fluorescence and NMR spectroscopy. Assembly of the protein into paired helical filaments after incubation with heparin at 37 °C was verified by electron microscopy and size‐exclusion chromatography. NMR spectroscopy on these mature fibers revealed different regions of residual mobility for tau: the N‐terminal domain was found to maintain solution‐like dynamics and was followed by a large domain of decreasing mobility; finally the core region was distinguished by a solid‐like character. Heteronuclear‐NOE data indicate that the decreasing mobility is due to both a slowing down of the rapid nanosecond movements and the introduction of slower movements that lead to exchange broadening. Fluorescence spectroscopy confirmed the presence of this rigid core, and some degree of protection from hydrogen exchange for those residues was observed. Hence, our data give a more precise picture of the dynamics of tau when it is integrated into mature filaments and should provide further understanding of the molecular processes that govern aggregation.

Role of the Arg123–Tyr166 Paired Helix of Apolipoprotein A-I in Lecithin:Cholesterol Acyltransferase Activation

The Arg123–Tyr166central and Ala190–Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) and apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight α-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 ± 0.33 (mean ± S.D.; n = 3), 0.054 ± 0.009 (p < 0.001 versusapoA-I), and 1.3 ± 0.32 (p = not significantversus apoA-I) nmol of cholesteryl ester/h/μm, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reducedV max with unaltered K m . Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) was very similar. In conclusion, although substitution of the Arg123–Tyr166 central or Ala190–Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL.

Studying the Natively Unfolded Neuronal Tau Protein by Solution NMR Spectroscopy

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimers diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.

Tau aggregation in Alzheimer's disease: what role for phosphorylation?

The crucial role of the neuronal Tau protein in microtubule stabilization and axonal transport suggests that too little or too much Tau might lead to neuronal dysfunction. The presence of a hyper-phosphorylated but non-aggregated molecule as a toxic species that might sequester normal Tau is discussed. We present recent in vitro results that might allow to dissect the role of individual phosphorylation sites on its structure and function. We also discuss in this review the role of phosphorylation for the aggregation of the neuronal Tau protein, and compare it to the aggregation induced by external poly-anions.

Dynamics of carbohydrate residues of α1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White

Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.