SUMO1 hinders α-Synuclein fibrillation by inducing structural compaction.

Small Ubiquitin-like Modifier 1 (SUMO1) is an essential protein for many cellular functions, including regulation, signaling, etc., achieved by a process known as SUMOylation, which involves covalent attachment of SUMO1 to target proteins. SUMO1 also regulates the function of several proteins via non-covalent interactions involving the hydrophobic patch in the target protein identified as SUMO Binding or Interacting Motif (SBM/SIM). Here, we demonstrate a crucial functional potential of SUMO1 mediated by its non-covalent interactions with α-Synuclein, a protein responsible for many neurodegenerative diseases called α-Synucleinopathies. SUMO1 hinders the fibrillation of α-Synuclein, an intrinsically disordered protein (IDP) that undergoes a transition to ß-structures during the fibrillation process. Using a plethora of biophysical techniques, we show that SUMO1 transiently binds to the N-terminus region of α-Synuclein non-covalently and causes structural compaction, which hinders the self-association process and thereby delays the fibrillation process. On the one hand, this study demonstrates an essential functional role of SUMO1 protein concerning neurodegeneration; it also illustrates the commonly stated mechanism that IDPs carry out multiple functions by structural adaptation to suit specific target proteins, on the other. Residue-level details about the SUMO1-α-Synuclein interaction obtained here also serve as a reliable approach for investigating the detailed mechanisms of IDP functions.

An efficient combination of BEST and NUS methods in multidimensional NMR spectroscopy for high throughput analysis of proteins.

Application of Non Uniform Sampling (NUS) along with Band-selective Excitation Short-Transient (BEST) NMR experiments has been demonstrated for obtaining the important residue-specific atomic level backbone chemical shift values in short durations of time. This application has been demonstrated with both well-folded (ubiquitin) and unfolded (α-synuclein) proteins alike. With this strategy, the experiments required for determining backbone chemical shifts can be performed very rapidly, i.e., in ∼2 hours of spectrometer time, and this data can be used to calculate the backbone folds of proteins using well established algorithms. This will be of great value for structural proteomic investigations on one hand, where the speed of structure determination is a limiting factor and for application in the study of slow kinetic processes involving proteins, such as fibrillization, on the other hand.

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates.

Cyclophilin catalyzes the ubiquitous process "peptidyl-prolyl cis-trans isomerization," which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

Backbone and side-chain resonance assignments of Plasmodium falciparum SUMO.

One of the most debilitating diseases Malaria, in its different forms, is caused by protozoan of Plasmodium species. Deadliest among these forms is the "cerebral malaria" which is afflicted upon by Plasmodium falciparum. Plasmodium adopts numerous strategies including various post-translational modifications (PTMs) to infect and survive in the human host. These PTMs have proven their critical requirement in the Plasmodium biology. Recently, sumoylation has been characterized as one of the important PTMs and many of its putative substrates have been identified in Plasmodium. Sumoylation is the covalent attachment of SUMO protein to the substrate protein, which is mediated by an enzyme cascade involving activating (E1), conjugating (E2), and ligating enzymes (E3). Here, we report resonance assignment for 1H, 13C and 15N of Plasmodium falciparum SUMO (Pf-SUMO) protein determined by various 2D and 3D heteronuclear NMR experiments along with predicted secondary structures.

NMR assignments of mitochondrial cyclophilin Cpr3 from Saccharomyces cerevisiae.

Cyclophilins regulate protein folding, transport and signalling through catalysis of proline isomerization, and are ubiquitously expressed in both prokaryotes and eukaryotes. Cpr3 is the yeast mitochondrial cyclophilin and it is structurally and biophysically uncharacterized so far. Yeast cyclophilin gene cpr3 is essential for the lactate metabolism. Here, we report (1)H, (13)C, and (15)N chemical shift assignments of Cpr3 protein determined by various 2D and 3D heteronuclear NMR experiments at pH 6.5, and temperature 298 K.