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The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.

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Post-translational modification by small ubiquitin-like modifier (SUMO) proteins has been implicated in the regulation of a variety of cellular events. The functions of sumoylation are often mediated by downstream effector proteins harboring SUMOinteracting motifs (SIMs) that are composed of a hydrophobic core and a stretch of acidic residues. MBD1-containing chromatin-associated factor 1 (MCAF1), a transcription repressor, interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3. We used NMR spectroscopy to solve the solution structure of the SIM of MCAF1 bound to SUMO-3. The hydrophobic core of the SIM forms a parallel ␤-sheet pairing with strand ␤2 of SUMO-3, whereas its C-terminal acidic stretch seems to mediate electrostatic interactions with a surface area formed by basic residues of SUMO-3. The significance of these electrostatic interactions was shown by mutations of both SUMO-3 and MCAF1. The present structural and biochemical data suggest that the acidic stretch of the SIM of MCAF1 plays an important role in the binding to SUMO-3. Small ubiquitin-like modifier (SUMO)2 proteins conjugate post-translationally with target proteins through a series of enzymatic reactions that resemble ubiquitination (1-5). In contrast to ubiquitination, which is largely involved in regulating the degradation of target proteins by proteasomes or lysosomes (6), sumoylation appears to regulate a wide variety of cellular events, such as nuclear transport, subnuclear localization, transcriptional regulation, DNA repair, and chromosome segregation (3). In particular, sumoylation of transcription factors represses transcription through a variety of different mechanisms, such as recruitment of histone deacetylases and regulation of nuclear body components (7). It has been suggested that sumoylation generally regulates the functions of target proteins by modulating their proteinprotein or protein-DNA interactions.In mammals, four SUMO paralogues, SUMO-1 through SUMO-4, have been identified, of which SUMO-1 to -3 can serve as protein modifiers (8). Whereas SUMO-2 and SUMO-3 share 97% amino acid identity, they have only 48 and 46% identity with SUMO-1, respectively. This indicates that SUMO-2 and SUMO-3 constitute a subgroup that is distinct from SUMO-1. Although these SUMO paralogues largely share common cellular functions, they also show paralogue-specific properties in regard to cellular localization and substrate specificity (9, 10). For example, SUMO-1 is preferentially found within nucleoli, nuclear envelopes, and cytoplasmic foci (8), whereas SUMO-2/3 accrue on chromosomes early in the nuclear reformation process (11). With respect to substrate specificity, RanGAP1 is preferentially modified by SUMO-1, whereas SUMO-2/3 show preference for topoisomerase 2 during mitosis. However, promyelocytic leukemia protein conjugates to all three SUMO paralogues (12). SUMO-1 is most abundant in the protein-conjugated form, whereas SUMO-2/3 isomers are more abundant in a free pool and are available to conjugate with target proteins...

The multidrug transporter AcrB actively exports a wide variety of noxious compounds using proton-motive force as an energy source in Gram-negative bacteria. AcrB adopts an asymmetric structure comprising three protomers with different conformations that are sequentially converted during drug export; these cyclic conformational changes during drug export are referred to as functional rotation. To investigate functional rotation driven by proton-motive force, all-atom molecular dynamics simulations were performed. Using different protonation states for the titratable residues in the middle of the transmembrane domain, our simulations revealed the correlation between the specific protonation states and the side-chain configurations. Changing the protonation state for Asp408 induced a spontaneous structural transition, which suggests that the proton translocation stoichiometry may be one proton per functional rotation cycle. Furthermore, our simulations demonstrate that alternating the protonation states in the transmembrane domain induces functional rotation in the porter domain, which is primarily responsible for drug transport.