Two-component dynamics of the order parameter of high temperature Bi2Sr2CaCu2O8+delta superconductors revealed by time-resolved Raman scattering.

We study the dynamics of the superconducting order parameter in the high-Tc cuprate Bi2Sr2CaCu2O8+delta by employing a novel time-resolved pump-probe Raman experiment. We find two different coupling mechanisms that contribute equally to the pair-breaking peak. One coupling sets in very fast at 2 ps and relaxes slowly, while the other one is delayed and sets in roughly at 5 ps and relaxes fast. A model that couples holes through phonons is able to reproduce one part of the condensate dynamics; thus, we argue that hole-spin interactions are of importance as well.

Coalescence of spherical beads of retro-HSP12.6 into linear and ring-shaped amyloid nanofibers.

The sequence-reversed form of a small heat shock protein, HSP12.6 (retro-HSP12.6), has been reported to fold and assemble into structured tetramers in aqueous solution. Upon raising the protein concentration to ~1.0-1.5 mg/ml, tetrameric retro-HSP12.6 is known to display a tendency to associate further into spherical beads of 18-20 nm in diameter containing folded protein subunits. Here we report that storage of this protein at low temperatures leads to further association of the beaded structures into linear and ring-shaped amyloid nanofibers of 18-20 nm in diameter. The electron micrographs presented in this communication provide the best visual evidence yet that amyloids can form through the association of smaller structured bead-like intermediates. The results also suggest that folded beta-sheet-rich subunits can participate in amyloid formation.

Gaplike excitations in the superconducting state of Bi2Sr2CaCu2O8 studied by resonant Raman scattering.

We report on the response of the electronic continuum from inelastic light-scattering experiments over an extended energy range between 1.970 and 4.504 eV in the superconducting state of Bi2Sr2CaCu2O8. The formation of a substantial Raman feature at shifts below twice the superconducting gap as well as the additional weight above this energy are found to be strongly dependent on the incident photon energy. For excitation wavelengths observed in ultraviolet, we find an enhancement of the integrated spectral weight below T(c). The resulting composite feature shows three distinct resonances at 2.5, 3.3, and 3.8 eV. We strongly suggest that the superconductivity-induced changes are the result of both the opening of a superconducting gap and the appearance of a collective mode.

A novel UV laser-induced visible blue radiation from protein crystals and aggregates: scattering artifacts or fluorescence transitions of peptide electrons delocalized through hydrogen bonding?

Proteins lacking prosthetic groups and/or cofactors are known to undergo electronic excitation transitions only upon exposure to UV-C (< 280 nm) and UV-B (280-320 nm), but not UV-A (320-400 nm) photons. Here, we report the discovery of a novel excitation that peaks at approximately 340 nm and yields visible violet-blue radiation with apparent band maxima at approximately 425, 445, 470, and 500 nm. All proteins and large polypeptides examined in solid form, and in solutions, display this quenchable and photobleachable radiation which can be established not owing to aromatic sidechains. As a note of caution, we wish to state that we have not been able to completely eliminate the possibility that the radiation may be an artifact owing to second order effects such as, e.g., Raman scattering of Raman-scattered photons; however, we assert that all our experiments indicate that the radiation actually owes to some form of fluorescence. We propose that peptide electrons that have been delocalized through intramolecular or intermolecular hydrogen bond formation display these long-wavelength electronic transitions. If confirmed by future studies, this preliminary discovery may turn out to have important implications for biomolecular spectroscopy, protein crystallography, and materials science.

Hydrophobic dye inhibits aggregation of molten carbonic anhydrase during thermal unfolding and refolding.

Association-seeking surfaces on partially structured polypeptides can participate in interactions that are either intramolecular (folding related) or intermolecular (aggregative). During heat shock, intermolecular associations leading to aggregation are prevented through the binding of such surfaces by chaperones of the Hsp20 family (with Hsp70 later effecting release and refolding). Here we report that the hydrophobic dye, 8-anilino-1-naphthalenesulfonate (ANS), mimics the function of the chaperones in its interactions with molten carbonic anhydrase (CA). At 150-fold molar excess of dye over protein, heat-induced aggregation of CA is almost completely inhibited by binding of ANS to solvent-exposed clusters of nonpolar residues. After exposure of ANS-containing protein solutions to temperatures as high as 95 degrees C, refolded CA can be recovered through cooling and dialysis, with no accompanying aggregation. This apparent mimicking of chaperone activity by a small dye opens up new approaches to understanding and manipulating protein aggregation.

Resolving multiple protein conformers in equilibrium unfolding reactions: a time-resolved emission spectroscopic (TRES) study of Azurin.

Unlike steady-state spectrofluorimetry, time-resolved emission spectroscopy (TRES) can resolve emissions from fluorophores with similar quantum yields and overlapping steady-state emission spectra. Time-resolved emission studies of the protein-intrinsic fluorophore, tryptophan (Trp), can thus potentially be used to examine protein conformational heterogeneity in solution, as well as to investigate the existence of populated intermediate structural states in equilibrium unfolding reactions of single-tryptophan proteins. Here, the single-Trp copper protein, azurin, is examined in various concentrations of guanidine hydrochloride (GdnCl) with its disulphide bond in an intact state. Interestingly, multiple envelopes of Trp emission are observed in all TRES spectra acquired, instead of just two emission envelopes (corresponding to the native and unfolded states) expected from two-state unfolding. These envelopes appear to be centred around the same set of emission wavelengths in different TRES spectra, and only intensities and decay rates vary with the concentration of denaturant used. This suggests that structural states representing different levels of exposure of Trp to the aqueous solvent might, in fact, be populated at equilibrium during the unfolding of azurin by GdnCl.

Cooperative relaxation of supercoils and periodic transcriptional initiation within polymerase batteries.

Transcription and DNA supercoiling are known to be linked by a cause-effect relationship that operates in both directions. It is proposed here that this two-way relationship may be exploited by the E. coli genome to facilitate constitutive transcription of supercoil-sensitive genes by polymerase batteries made up of uniformly spaces RNA polymerase elongation complexes. Specifically, it is argued that (1) polymerases transcribing DNA in tandem cooperate to relax each other's transcription-driven positive supercoils; and (2) negative supercoils driven upstream by elongation complexes tend to be 'harnessed' and used to cooperatively (and periodically) initiate fresh transcription from promoters. Harnessing of transcription-driven negative supercoils is thought to be achieved through the erection of protein barriers to the rotational upstream propagation of supercoils from transcription events. The possible relevance of such cooperation amongst polymerases to the activation of transcription by DNA-binding protein factors is emphasized. Some testable predictions are made and implications are discussed.

Does replication-induced transcription regulate synthesis of the myriad low copy number proteins of Escherichia coli?

Over 80% of the genes in the E. coli chromosome express fewer than a hundred copies each of their protein products per cell. It is argued here that transcription of these genes is neither constitutive nor regulated by protein factors, but rather, induced by the act of replication. The utility of such replication-induced (RI) transcription to the temporal regulation of synthesis of determinate quantities of low copy number (LCN) proteins is described. It is suggested that RI transcription may be necessitated, as well as facilitated, by the folding of the bacterial chromosome into a compact nucleoid. Mechanistic aspects of the induction of transcription by replication are discussed with respect to the modulation of transcriptional initiation by negative supercoiling effects, promoter methylation status and derepression. It is shown that RI transcription offers plausible explanations for the constancy of the C period of the E. coli cell cycle and the remarkable conservation of gene order in the chromosomes of enteric bacteria. Some experimental tests of the hypothesis are proposed.

Dityrosine formation in the proteins of the eye lens.

The presence of dityrosine crosslinks in the proteins of the lens is a subject of some debate. We have investigated the formation of dityrosine in the lens proteins, the crystallins, through reactions mediated by reactive oxygen species, as well as through direct photolysis of proteins in the UVB region. Multiple methods were used to identify dityrosine. These include amino acid analysis of protein hydrolysates, quenching of the fluorescence at 400-410 nm by borate/boric acid solutions specific for dityrosine, and inhibition of formation of dityrosine in the presence of dithiothreitol. We find that reaction of the crystallins, alpha (alpha), beta (beta) and gamma (gamma), with singlet oxygen (1O2), hydroxyl radicals (.OH) and hydrogen peroxide (H2O2), does not result in the formation of either intermolecular or intramolecular dityrosine. It appears that the formation of dityrosine involves a radical mediated electron transfer reaction. Consistent with this, direct photolysis in the UVB region produces intramolecular dityrosine in all three proteins, with the efficiency varying in the order gamma - > beta - > alpha. Since the levels of electron transfer agents (e.g., Fe ions) in the lens are usually negligible, and since the amount of UVB radiation reaching the gamma--crystallin--rich lens nucleus is normally quite low, the changes of dityrosine formation in the crystallins in vivo appear remote.

Reversal of peptide backbone direction may result in the mirroring of protein structure.

In linear polypeptides, inversion of amino acid chirality (all-L to all-D) achieves a mirroring of side chain positions and interactions in conformational space. A similar mirroring of side chain positions is independently achieved by a reversal of the direction of the peptide backbone (retro modification). Thus, while an all-D chain could be expected to adopt a perfect 'mirror image' of the three-dimensional structure of its parent all-L protein, the retro-all-L chain could be expected to adopt a topological equivalent of such a mirror image, through the symmetry transformations of side chain interactions. These notions, supported by sequence analyses, modelling studies, and evidence relating to the activity of 'retro-inverso' peptides, are extended towards the proposal, that the backbone reversed chain of a large globular protein might recognize the chiral opposite of the parent protein's substrate(s).

Hydroxyl radical mediated damage to proteins, with special reference to the crystallins.

Oxidative modification of the eye lens proteins, the crystallins, is known to cause protein cross-links and aggregates which lead to lens opacification or cataracts. We focus attention here on oxidative damage occurring in crystallins and some "control" proteins upon reaction with the hydroxyl radical (.OH) which, in the lens, is generated by photosensitization or by the Fenton reaction. In the present study, we have synthesized and used the bishydroperoxide I as a "photo-Fenton" reagent, in order to photolytically generate pure .OH, free of other oxyradicals. Our findings are the following: (i) Trp residues are oxidized by .OH to N-formylkynurenine and related compounds, but this in itself does not lead to covalent aggregation of the protein. (ii) Tyr residues react with .OH, but apparently do not produce dihydroxyphenylalanine or bityrosine. Nor do protein cross-links occur as a result. (iii) Oxidation of His residues appears to be obligatory for protein cross-linking. Histidine-free proteins do not form high molecular weight products upon reaction with .OH. Protection of His residues by adduct formation in other proteins inhibits cross-linking. (iv) Lys residues seem to participate in the cross-linking reaction. Protection of the Lys residues by maleylation of the protein inhibits cross-linking. (v) The oxidized protein is more acidic in nature than the parent, and it might have altered conformational features.