Spatial Extent of Charge Repulsion Regulates Assembly Pathways for Lysozyme Amyloid Fibrils

Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path

Kinetics and mechanism of the photochemical reaction of 2,2 '-dipyridyl with tryptophan in water: Time-resolved CIDNP and laser flash photolysis study

The mechanism of the reactions between photoexcited 2,2′-dipyridyl and N-acetyl tryptophan has been studied by laser flash photolysis and time-resolved CIDNP (Chemically Induced Dynamic Nuclear Polarization). The transient absorption spectra obtained at different delays after the laser pulse are attributed to the triplet state of dipyridyl and to dipyridyl and tryptophan radicals. Depending on the pH of the solution, all three intermediates can be present in either protonated or deprotonated forms. It is shown that irrespective of pH the primary photochemical step is electron transfer from the tryptophan to triplet dipyridyl followed by protonation/deprotonation of the radicals so formed. The rate constant of the reaction of triplet dipyridyl with tryptophan is close to the diffusion-controlled limit and decreases slightly with increasing pH. The kinetics and the stationary value of the CIDNP are determined by the rates of radical termination, nuclear paramagnetic relaxation, and degenerate electron exchange. The last reaction is important for the protonated tryptophan radical and determines the CIDNP kinetics of tryptophan in acidic conditions. The nuclear relaxation times estimated from the CIDNP kinetics are 44 ± 9 μs for all protons in the dipyridyl radical, 91 ± 20 μs for the β-CH2, 44 ± 9 μs for H2,6, and 63 ± 12 μs for H4 aromatic protons in the tryptophan radical

Intramolecular electron transfer in lysozyme studied by time-resolved chemically induced dynamic nuclear polarization.

The kinetics of the chemically induced dynamic nuclear polarization (CIDNP) produced in reactions of hen lysozyme with photosensitizers have been studied for the native state of the protein at pH 3.8 and for two denatured states. The latter were generated by raising the temperature to 80 degrees C or by combining a temperature rise (to 50 degrees C) with the addition of chemical denaturant (10 M urea). Detailed analysis of the CIDNP time dependence on a microsecond time scale revealed that, in both denatured states, intramolecular electron transfer (IET) from a tyrosine residue to the cation radical of a tryptophan residue (rate constant k(f)) is highly efficient and plays a decisive role in the evolution of the nuclear polarization. To describe the observed CIDNP kinetics with a self-consistent set of parameters, IET in the reverse direction, from a tryptophan residue to a tyrosine residue radical (rate constant k(r)), has also to be taken into account. The IET rate constants determined by analysis of the CIDNP kinetics are, at 80 degrees C: k(f) = 1 x 10(5) s(-1) and k(r) = 1 x 10(4) s(-1); at 50 degrees C in the presence of 10 M urea: k(f) = 7 x 10(4) s(-1), k(r) = 1 x 10(4) s(-1). IET does not appear to influence the CIDNP kinetics of the native state

Time-resolved CIDNP study of non-native states of bovine and human alpha-lactalbumins.

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of the aromatic dye 2,2'-dipyridyl with non-native states of bovine and human alpha-lactalbumins (BLA and HLA) in aqueous solution have been studied using the time-resolved CIDNP technique. Non-native states have been obtained at pH 2 in the presence of 0, 8, and 10 M urea-d(4) and at pH 6.7 in the presence of 10 M urea-d(4). The dependence of the geminate CIDNP spectra of the two proteins on the denaturant concentration is shown to be determined by the intrinsic reactivity of the amino acid residues toward the triplet excited dye rather than by structural changes in the proteins. Values of the proton paramagnetic relaxation times (T(1)) have been obtained from an analysis of the CIDNP kinetics. For tryptophan and tyrosine residues, the T(1) values change in opposite directions when the proteins are progressively denatured, reflecting the different internal mobilities of the two types of residues. It has been found that for both BLA and HLA the CIDNP kinetics of the non-native states formed at pH 6.7 in the presence of 10 M urea are almost identical to those at pH 2 with no urea, suggesting that the polarizable amino acid side chains have closely similar solvent accessibilities and motional properties in the two non-native states

Time-resolved CIDNP study of native-state bovine and human alpha-lactalbumins

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of two aromatic dyes (2,2′-dipyridyl, DP, and flavin mononucleotide, FMN) with bovine and human α-lactalbumins (BLA and HLA) in aqueous solution have been studied using the time-resolved CIDNP technique. With DP as the photosensitizer, polarization in BLA is observed for the protons of Trp118, His68, Tyr18, and Tyr103. The latter is not manifested in the CIDNP spectra obtained with FMN. This threshold effect on CIDNP formation indicates that accessibility is a combined property of both the residue and the dye. The nuclear spin-lattice relaxation times in the radicals formed in these reactions have been determined from the CIDNP kinetics: T1 = 60 μs for H2,4,7 of Trp118, T 1 = 53 μs, for H3,5 of Tyr103, T1 = 16 μs for H3,5 of Tyr18, and T1 = 10 μs for the H2 and H4 of His68. The correlation times for the side chain motion as determined from the T1 of the radicals are correlated with the accessibility of the side chains in the intact protein

1H and 13C hyperfine coupling constants of the tryptophanyl cation radical in aqueous solution from microsecond time-resolved CIDNP.

Relative values of the 1H and 13C isotropic hyperfine couplings in the cationic oxidized tryptophan radical TrpH*+ in aqueous solution are determined. The data are obtained from the photo-CIDNP (chemically induced dynamic nuclear polarization) enhancements observed in the microsecond time-resolved NMR spectra of the diamagnetic products of photochemical reactions in which TrpH*+ is a transient intermediate. The method is validated using the tyrosyl neutral radical Tyr*, whose 1H and 13C hyperfine couplings have previously been determined by electron paramagnetic resonance spectroscopy. Good agreement is found with hyperfine coupling constants for TrpH*+ calculated using density functional theory methods but only if water molecules are explicitly included in the calculation

Time-resolved CIDNP and laser flash photolysis study of the photoreactions of N-acetyl histidine with 2,2 '-dipyridyl in aqueous solution

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of 2,2′-dipyridyl (DP) and N-acetyl histidine (HisH) in aqueous solution have been studied using laser flash photolysis and time-resolved CIDNP techniques. The triplet state TDP reacts with protonated HisH 2+ via hydrogen atom transfer with a rate constant k H = 1.2 × 10 8 M -1 s -1, and with deprotonated His - via electron transfer with k e = 7.5 × 10 9 M -1 s -1. No reaction occurs when the histidine imidazole ring is in its neutral state HisH, or when the dipyridyl triplet is protonated, TDPH +. The nuclear spin-lattice relaxation times in the radicals formed in these reactions have been determined from the CIDNP kinetics: T 1 = 44 ± 9 μs for all DP protons, T 1 = 196 ± 25 μs for the β-CH 2 protons of HisH, and T 1 = 16 ± 5 μs for the H-2 and H-4 protons of HisH. Under strongly basic conditions the CIDNP is greatly affected by degenerate electron exchange between the neutral His • radical and His - anion, with rate constant k ex = 1.5 × 10 8 M -1 S -1. © 2000 American Chemical Society

Time resolved CIDNP study of electron transfer reactions in proteins and model compounds

Intramolecular electron transfer (IET) from tyrosine to tryptophan cation radicals is investigated using time resolved chemically induced dynamic nuclear polarization (CIDNP) spectroscopy in combination with laser flash photolysis. In both the tryptophan-tyrosine dipeptide and the denatured state of hen lysozyme in aqueous solution, the transformation TrpH+. → TyrO. by IET leads to an increase in the tyrosine radical concentration, growth in the tyrosine CIDNP signal, fast decay of the tryptophan CIDNP, and inversion of the phase of the CIDNP of the photosensitizing dye, 2,2′-dipyridyl. IET effects are not observed for mixtures of the amino acid or for the native state of lysozyme. The steady state CIDNP effects seen for denatured lysozyme thus depend not only on the accessibility of the amino acid residues on the surface of the protein but also on the reactivity of the radical intermediates

Effects of surfactants on the photosensitized production of tyrosine radicals studied by photo-CIDNP.

The influence of the surfactants sodium dodecyl sulphate, cetyltrimethyl-ammonium bromide and triton X-100 on the photochemically induced dynamic nuclear polarization (CIDNP) of N-acetyl tyrosine has been investigated. Three photosensitizers were used to generate polarization: thionin, eosin Y and flavin mononucleotide. 600 MHz 1H photo-CIDNP experiments, supported by laser flash photolysis transient absorption measurements, indicate that the neutral triton surfactant has no influence on the nuclear polarization, but that the other two, charged, amphiphiles affect the photochemistry in a variety of ways, depending on the surfactant concentration and the identity of the sensitizer