The interactions of several water-soluble ionic porphyrins with different ionic or neutral surfactants in aqueous solutions were studied as a function of surfactant concentration. The interaction leads to the formation of porphyrin aggregates and/or micelle-encapsulated monomers with the exception of those porphyrin−surfactant pairs for which the interaction is Coulombically repulsive. The premicellar surfactant−porphyrin aggregate is identified by absorption and fluorescence spectroscopy, fluorescence lifetime and anisotropy, and resonance light scattering. The spectroscopic results are used to characterize the premicellar aggregates as J-type, H-type, or nonspecific aggregates. All premicellar surfactant−porphyrin aggregates dissociate to form micelle-encapsulated monomers when the surfactant concentration approaches cmc (critical micellar concentration). The interaction of tetrakis-(4-sulfanatophenyl)porphine dianion (H4TPPS2-) at pH <3.5 with cetyltrimethylammonium cation (CTAB) is described by the following sequential equlibria controlled by the surfactant concentration:  M ⇌ J ⇌ H ⇌ Mm. The stoichiometric ratio of porphyrin/surfactant is 1:2 for the J-aggregate and ∼1:4 for the H-aggregate. Kinetic intermediates were also observed prior to the formation of the J-aggregate. The J-aggregate exhibits circular dichroism (spontaneous chirality, not seen in H-type or micellar aggregates), intense resonance light scattering, low fluorescence quantum yield and lifetime, and unusually high fluorescence anisotropy.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

The fluorescence depolarization dynamics of organic fluorescent dye probes (nile red, cresyl violet, DODCI, rhodamine B, and rhodamine DPPE) were studied in cationic, anionic, and neutral micelles by picosecond time-resolved single-photon-counting technique. The fluorescence anisotropy decay of the dye intercalated inside the micelle is a two-exponential function. The anisotropy decay was interpreted by using a model of rotational (wobbling) and translational diffusion of the dye in the micelle coupled with the rotational motion of the micelle as a whole. The rotational and translational diffusion coefficients of the dye, the order parameter, and the semicone angle for the wobbling diffusion in the micelle were determined. The concept of “microviscosity” in the micelle was critically discussed in the light of the rotational and translational diffusion coefficients and their temperature dependence.

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Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.

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The zinc-dependent matrix metalloproteinases (MMPs) are key enzymes associated with extracellular matrix (ECM) remodeling; they play critical roles under both physiological and pathological conditions. MMP-9 activity is linked to many pathological processes, including rheumatoid arthritis, atherosclerosis, gastric ulcer, tumor growth, and cancer metastasis. Specific inhibition of MMP-9 activity may be a promising target for therapy for diseases characterized by dysregulated ECM turnover. Potent MMP-9 inhibitors including an indole scaffold were recently reported in an X-ray crystallographic study. Herein, we addressed whether melatonin, a secretory product of pineal gland, has an inhibitory effect on MMP-9 function. Gelatin zymographic analysis showed a significant reduction in pro- and active MMP-9 activity in vitro in a dose- and time-dependent manner. In addition, a human gastric adenocarcinoma cell line (AGS) exhibited a reduced (~50%) MMP-9 expression when incubated with melatonin, supporting an inhibitory effect of melatonin on MMP-9. Atomic-level interaction between melatonin and MMP-9 was probed with computational chemistry tools. Melatonin docked into the active site cleft of MMP-9 and interacted with key catalytic site residues including the three histidines that form the coordination complex with the catalytic zinc as well as proline 421 and alanine 191. We hypothesize that under physiological conditions, tight binding of melatonin in the active site might be involved in reducing the catalytic activity of MMP-9. This finding could provide a novel approach to physical docking of biomolecules to the catalytic site of MMPs, which inhibits this protease, to arrest MMP-9-mediated inflammatory signals.