@article {78107, title = {Monitoring protein ubiquitination and SUMOylation in real-time by NMR.}, journal = {Chem Commun (Camb)}, volume = {56}, year = {2020}, month = {2020 Jun 18}, pages = {6735-6738}, abstract = {

The covalent conjugation of ubiquitin (Ub), known as ubiquitination, is a multi-step reaction involving multiple enzymes. We report a real-time, tag-free method to monitor protein ubiquitination by NMR spectroscopy under physiological conditions. The approach is also applicable for monitoring other ubiquitin-like modifications, and the disassembly of Ub polymers.

}, issn = {1364-548X}, doi = {10.1039/d0cc02252g}, author = {Habibullah, Batul Ismail and Tripathi, Vasvi and Surana, Parag and Das, Ranabir} } @article {50995, title = {A five-residue motif for the design of domain swapping in proteins.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 28}, pages = {452}, abstract = {

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue {\textquoteright}cystatin motif{\textquoteright} (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08295-x}, author = {Nandwani, Neha and Surana, Parag and Negi, Hitendra and Mascarenhas, Nahren M and Udgaonkar, Jayant B and Das, Ranabir and Gosavi, Shachi} } @article {20316, title = {Amino-acid composition after loop deletion drives domain swapping.}, journal = {Protein Sci}, volume = {26}, year = {2017}, month = {10/2017}, chapter = {1994}, abstract = {

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain-swapped oligomer over the monomer. While it is known that the deletion of loops between β-strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop-deletion mutants of the non-domain-swapping protein monellin, frame-shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.3237}, author = {Nandwani, Neha and Surana, Parag and Udgaonkar, Jayant B and Das, Ranabir and Gosavi, Shachi} } @article {24895, title = {Structural and functional analysis of SMO-1, the SUMO homolog in Caenorhabditis elegans.}, journal = {PLoS One}, volume = {12}, year = {2017}, month = {2017}, pages = {e0186622}, abstract = {

SUMO proteins are important post-translational modifiers involved in multiple cellular pathways in eukaryotes, especially during the different developmental stages in multicellular organisms. The nematode C. elegans is a well known model system for studying metazoan development and has a single SUMO homolog, SMO-1. Interestingly, SMO-1 modification is linked to embryogenesis and development in the nematode. However, high-resolution information about SMO-1 and the mechanism of its conjugation is lacking. In this work, we report the high-resolution three dimensional structure of SMO-1 solved by NMR spectroscopy. SMO-1 has flexible N-terminal and C-terminal tails on either side of a rigid beta-grasp folded core. While the sequence of SMO-1 is more similar to SUMO1, the electrostatic surface features of SMO-1 resemble more with SUMO2/3. SMO-1 can bind to typical SUMO Interacting Motifs (SIMs). SMO-1 can also conjugate to a typical SUMOylation consensus site as well as to its natural substrate HMR-1. Poly-SMO-1 chains were observed in-vitro even though SMO-1 lacks any consensus SUMOylation site. Typical deSUMOylation enzymes like Senp2 can cleave the poly-SMO-1 chains. Despite being a single gene, the SMO-1 structure allows it to function in a large repertoire of signaling pathways involving SUMO in C. elegans. Structural and functional features of SMO-1 studies described here will be useful to understand its role in development.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0186622}, author = {Surana, Parag and Gowda, Chandrakala M and Tripathi, Vasvi and Broday, Limor and Das, Ranabir} } @article {5305, title = {Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR.}, journal = {Protein Sci}, year = {2016}, month = {2016 Apr 25}, abstract = {

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by Nuclear Magnetic Resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with co-factors as well as for assembly of poly-Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein{\textquoteright}s core, the long loop converges on the core in the final step of the folding process. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.2940}, author = {Surana, Parag and Das, Ranabir} } @article {897, title = {Fast native-SAD phasing for routine macromolecular structure determination.}, journal = {Nat Methods}, volume = {12}, year = {2015}, month = {2015 Feb}, pages = {131-3}, abstract = {

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

}, keywords = {Animals, DNA-Binding Proteins, Humans, Membrane Proteins, Models, Molecular, Multiprotein Complexes, Protein Conformation, Software, Synchrotrons, X-Ray Diffraction}, issn = {1548-7105}, doi = {10.1038/nmeth.3211}, author = {Weinert, Tobias and Olieric, Vincent and Waltersperger, Sandro and Panepucci, Ezequiel and Chen, Lirong and Zhang, Hua and Zhou, Dayong and Rose, John and Ebihara, Akio and Kuramitsu, Seiki and Li, Dianfan and Howe, Nicole and Schnapp, Gisela and Pautsch, Alexander and Bargsten, Katja and Prota, Andrea E and Surana, Parag and Kottur, Jithesh and Nair, Deepak T and Basilico, Federica and Cecatiello, Valentina and Pasqualato, Sebastiano and Boland, Andreas and Weichenrieder, Oliver and Wang, Bi-Cheng and Steinmetz, Michel O and Caffrey, Martin and Wang, Meitian} }