Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, represent important intermediates in both physiological protein folding and pathological protein misfolding. To aid in the characterization of the structural state(s) of such proteins, a novel structure calculation scheme is presented that combines structural restraints derived from pulsed EPR and NMR spectroscopy. The methodology is established for the protein α-synuclein (αS), which exhibits characteristics of a partially folded protein when bound to a micelle of the detergent sodium lauroyl sarcosinate (SLAS). By combining 18 EPR-derived interelectron spin label distance distributions with NMR-based secondary structure definitions and bond vector restraints, interelectron distances were correlated and a set of theoretical ensemble basis populations was calculated. A minimal set of basis structures, representing the partially folded state of SLAS-bound αS, was subsequently derived by back-calculating correlated distance distributions. A surprising variety of well-defined protein-micelle interactions was thus revealed in which the micelle is engulfed by two differently arranged anti-parallel αS helices. The methodology further provided the population ratios between dominant ensemble structural states, whereas limitation in obtainable structural resolution arose from spin label flexibility and residual uncertainties in secondary structure definitions. To advance the understanding of protein-micelle interactions, the present study concludes by showing that, in marked contrast to secondary structure stability, helix dynamics of SLAS-bound αS correlate with the degree of protein-induced departures from free micelle dimensions.

Proteins that cap the ends of the actin filament are essential regulators of cytoskeleton dynamics. While several proteins cap the rapidly-growing barbed end, tropomodulin (Tmod) is the only protein known to cap the slowly-growing pointed end. The lack of structural information severely limits our understanding of Tmod’s capping mechanism. We describe crystal structures of actin complexes with the unstructured N-terminal and the leucine-rich repeat C-terminal domains of Tmod. The structures and biochemical analysis of structure-inspired mutants showed that one Tmod molecule interacts with three actin subunits at the pointed end, while also contacting two tropomyosin molecules on each side of the filament. We found Tmod achieves high affinity binding through several discrete low-affinity interactions, which suggests a mechanism for controlled subunit exchange at the pointed end.

The 140-residue protein alpha-synuclein (aS) has been implicated in the molecular chain of events leading to Parkinson's disease, which relates to the hierarchical aggregation of aS into soluble oligomers and insoluble fibrils. A number of small organic molecules have been reported to inhibit aS aggregation. Here, the interactions of chlorazole black E, Congo red, lacmoid, PcTS-Cu (2+), and rosmarinic acid with aS are examined by NMR spectroscopy to identify aS sequence elements that are masked by these compounds. Surprisingly, similar aS interaction sites, encompassing residues 3-18 and 38-51, were obtained for all molecules at equimolar small molecule:aS ratios. At higher ratios, virtually the entire amphiphilic region of aS (residues 2-92) is affected, revealing the presence of additional, lower affinity interaction sites. Upon rearranging the high-affinity interaction sites over the aS amphiphilic region in an aS mutant form, perturbations of the entire amphiphilic region were found to have already been obtained at equimolar ratios, indicating a high specificity for the original binding sites. CD spectroscopy reveals that, in the presence of the small molecules, the aS structure is still dominated by random-coil characteristics. The strongest effects are exerted by molecules that contain sulfonate groups adjacent to aromatic systems, often present in multiple copies in a symmetrical arrangement, suggesting that these elements are useful for developing an aS-specific chemical chaperone.

The pathological and physiological hallmarks of the protein α-synuclein are its misfolding into cytotoxic aggregates and its binding to synaptic vesicles, respectively. Both events are mediated by seven 11-residue amphiphilic pseudorepeats and, most generally, involve a transition from intrinsically unstructured to structured conformations. Based upon α-synuclein interactions with aggregation-inhibiting small molecules, a α-synuclein variant termed SaS, wherein the first six pseudorepeats had been rearranged, was introduced. Here, the effects of this rearrangement upon misfolding, vesicle binding and micelle binding are examined in reference to α- and β-synuclein to study the sequence characteristics underlying these processes. Fibrillization correlates with the distinct clustering of residues with high β-sheet propensities, while vesicle affinities depend on the mode of pseudorepeat interchange and loss. In the presence of micelles, the pseudorepeat region of SaS adopts an essentially continuous helix, whereas α- and β-synuclein encounter a distinct helix break, indicating that a more homogeneous distribution of surfactant affinities in SaS prevented the formation of a helix break in the micelle-bound state. By demonstrating the importance of the distribution of β-sheet propensities and by revealing inhomogeneous aS surfactant affinities, the present study provides novel insight into two central themes of synuclein biology.

Background:The enzyme carnitine palmitoyltransferase 1 regulates the rate of fatty acid oxidation. Results: The regulatory domain of the enzyme constitutes an amphiphilic structural switch. Conclusion:The switch can integrate enzyme inhibitor concentration and membrane characteristics into one regulatory signal. Significance: This represents a highly sophisticated regulatory mechanism for enzymes residing at the membrane-water interface.

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.

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The E subunit of the human heterotetrameric negative transcription elongation factor (NELF-E) contains a canonical betaalphabetabetaalphabeta RNA recognition motif (RRM) that binds to a wide variety of RNA sequences. These induce very similar conformational changes in the RRM as determined by nuclear magnetic resonance spectroscopy. Although the RNA binding interface of a canonical RRM is mainly located at its beta-sheet surface, for NELF-E RRM large chemical shift perturbations are observed for residues in the flexible C-terminal region and the loop between beta 3 and alpha 2, and both regions are distant from the interface. We determined the solution structure of single-stranded transactivator responsive element (TAR) RNA-bound NELF-E RRM. This structure clearly shows that RNA binding to NELF-E RRM induces formation of a helix in the C-terminus. The RNA-bound form of NELF-E RRM is very similar to the RNA-bound form of U1A RRM, although the C-terminus of the NELF-E RRM is unstructured in the free protein, whereas it is helical in the U1A protein. Thus, RNA binding to NELF-E RRM induces a conformational change toward the U1A structure, resulting in highly similar RNA binding conformations for both proteins.