Olga Tcherkasskaya

Denatured collapsed states in protein folding: Example of apomyoglobin †

Experimental approaches, including circular dichroism, small angle X‐ray scattering, steady‐state fluorescence, and fluorescence energy transfer, were applied to study the 3D‐structure of apomyolgobin in different conformational states. These included the native and molten globules, along with either less ordered conformations induced by the addition of anions or completely unfolded states. The results show that the partially folded forms of apomyoglobin stabilized by KCl and/or Na2SO4 under unfolding conditions (pH 2) exhibit a significant amount of secondary structure (circular dichroism), low packing density of protein molecules (SAXS), and native‐like dimensions of the AGH core (fluorescence energy transfer). This finding indicates that a native‐like tertiary fold of the polypeptide chain, i.e., the spatial organization of secondary structure elements, most likely emerges prior to the formation of the molten globule state. Proteins 2001;44:244–254.

Local Flexibility in Molecular Function Paradigm

It is generally accepted that the functional activity of biological macromolecules requires tightly packed three-dimensional structures. Recent theoretical and experimental evidence indicates, however, the importance of molecular flexibility for the proper functioning of some proteins. We examined high resolution structures of proteins in various functional categories with respect to the secondary structure assessment. The latter was considered as a characteristic of the inherent flexibility of a polypeptide chain. We found that the proteins in functionally competent conformational states might be comprised of 20–70% flexible residues. For instance, proteins involved in gene regulation, e.g. transcription factors, are on average largely disordered molecules with over 60% of amino acids residing in “coiled” configurations. In contrast, oxygen transporters constitute a class of relatively rigid molecules with only 30% of residues being locally flexible. Phylogenic comparison of a large number of protein families with respect to the propagation of secondary structure illuminates the growing role of the local flexibility in organisms of greater complexity. Furthermore the local flexibility in protein molecules appears to be dependent on the molecular confinement and is essentially larger in extracellular proteins.

The role of hydrophobic interactions in amyloidogenesis: Example of prion-related polypeptides

Abstract Conversion of the non-infectious, cellular forom of the prion protein (PrPC) to the infectious form (PrPSc) is thought to be driven by an α-helical to β-sheet conformational transition. To reveal the sequence determinants which encourage the transition to (β-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and sleady stale fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant β-strand formation, i.e., before the appearance of any ordered secondary structure delectable by circular dichroism. At moderate concentrations of trifluo-roethanol and/or acetonitrile, the conformational ensemble shifts towards β-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid (β-sheet structures. Encouragement of β-folds requires the deslabilizalion of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms

Femtosecond Fluorescence Spectra of Tryptophan in Human γ-Crystallin Mutants: Site-Dependent Ultrafast Quenching

The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.

Molten globule versus variety of intermediates: influence of anions on pH-denatured apomyoglobin

The molten globule state was shown to be the third thermodynamic state of protein molecules in addition to their native and unfolded states. On the other hand, it was reported that optical and hydrodynamic properties of pH‐denatured apomyoglobin depend on the nature of anions added to the protein solution. This observation was used to conclude that there are many ‘partly folded’ intermediates between the native and unfolded states rather than one distinct molten globule state. However, little is known on the structures of pH‐denatured apomyoglobin in the presence of different anions. Two tyrosine residues in horse apomyoglobin have been successively modified by the reaction with tetranitromethane. This approach was employed to measure the distances between tryptophans and modified tyrosines in different states of apomyoglobin by the method of direct energy transfer. Experimental data show that the distance between the middle of the A‐helix and the beginning of the G‐helix and/or the end of the H‐helix in ‘anion‐induced’ states are very close to those in the native holo‐ and apomyoglobins. This suggests that the AGH helical complex, being the most structured part of apomyoglobin in the molten globule state, exists also in pH‐denatured apomyoglobin in the presence of different anions. Consequently, all non‐native forms of apomyoglobin studied so far share the common important feature of its native structure.

Polymeric aspects of protein folding: a brief overview.

Regardless of the differences in primary amino acid sequences, protein molecules in a number of conformational states behave as polymer homologues, allowing speculations as to the volume interactions being a driving force in formation of equilibrium structures. For instance, both native and molten globules exhibit key features of polymer globules, where the fluctuations of the molecular density are expected to be much less than the molecular density itself. Protein molecules in the compact denatured (pre-molten globule) states possess properties of squeezed coils. In fact, even high concentrations of strong denaturants (e.g., urea and GdmCl) more likely constitute bad solvents for protein chains. Thus, globular proteins are probably never random coils without positional correlations and biological polypeptide chains represent the macromolecular coils below a critical point even under harsh denaturing conditions. Several implications of these findings to protein folding are discussed.

Photo‐activity induced by amyloidogenesis

Accumulation of chemically altered proteins is a noted characteristic of biological aging, and increasing evidence suggests a variety of deleterious cellular developments associated with senescence. Concomitantly, the “aging” of protein deposits associated with numerous neurological disorders may involve covalent modifications of their constituents. However, the link between disease‐related protein aggregation and chemical alterations of its molecular constituents has yet to be established. The present study of amyloidogenic α‐synuclein protein points to a decisive change in the biophysical behavior of growing protein aggregates with progressive photo‐activity in the visible range of the electromagnetic spectrum. I hypothesize that the photo‐activity induced by filament formation is governed by the same mechanism as seen for the intrinsic chromophore of 4‐(p‐hydroxybenzylidene)‐5‐imidazolinone‐type in the family of green fluorescent proteins. This type of the covalent alterations is initiated concurrently with amyloid elongation and involves a complex multi‐step process of chain cyclization, amino acid dehydration, and aerial oxidation. Given that different stages in filament formation yield distinct optical characteristics, the photo‐activity induced by amyloidogenesis may have application in molecular biology by enabling in vivo visualization of protein aggregation and its impact on cellular function.

Effects of molecular distribution on the fluorescence transfer: Exact results for slab geometry

Fluorescence energy transfer between donor and acceptor molecules in nonuniform molecular distributions is considered. In particular, we investigate the effect of the molecular distributions on the time-resolved fluorescence intensity decay of the donor and provide exact analytical expressions for the ensemble-averaged donor decays when the acceptor molecules are distributed uniformly within a slab of a finite thickness. The effect of the donor distribution on the energy transfer is also examined. Importantly, the derived formulas display both asymptotic limits for the two- and three-dimensional molecular distributions, and the crossover between them. Analytical results show very good agreement with numerical lattice model simulations. On this basis, we introduce new procedures for the energy transfer data analysis that provide a direct estimation of the slab thickness and the acceptor concentration from a single time-resolved fluorescence measurement.

Is 10-100ps Spectral Relaxation of Trp An Indicator of Local Disorder in Proteins?

We have studied the time-resolved fluorescence of the Trp43 residue of the globular protein GB1 upon acid induced equilibrium unfolding.NMR structural experiments have shown this protein is actually very acid stable above pH3.Nanosecond time-resolved TCSPC data clearly suggest that, in the tryptophan environment, partial unfolding appears at surprisingly high pH values. In fact, GB1 exhibits signs of local lifetime changes for pH values as high as 6.9.Further, femtosecond ultraviolet upconversion data reveal a ∼30 ps component with a negative preexponential amplitude in the longer wavelength portion of the emission spectrum. Such a rise time on the red side of emission is a signature of generalized relaxation (solvent and/or protein) around the excited dipole of Trp. A similar term with a ∼2ps exponential is always found for proteins in aqueous solution, representing bulk water motion. Slower terms (10-100ps) have previously been assigned to unusual water environments, protein dipolar relaxation, or the coupling between them.Most intriguing is the fact that, for somewhat lower pH values where GB1 is locally (but not globally) unfolded to a larger degree, the amplitude of the observed 30ps term becomes larger (more negative).Femtosecond Trp emission spectroscopy may thus provide new snapshots of proteins that are “fully folded” over longer time averaging but still have transiently unstructured regions.