%0 Journal Article %J RSC Adv %D 2022 %T Modulation of biliverdin dynamics and spectral properties by Sandercyanin. %A Ghosh, Swagatha %A Mondal, Sayan %A Yadav, Keerti %A Aggarwal, Shantanu %A Schaefer, Wayne F %A Narayana, Chandrabhas %A Subramanian, Ramaswamy %X

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

%B RSC Adv %V 12 %P 20296-20304 %8 2022 Jul 06 %G eng %N 31 %R 10.1039/d2ra02880h %0 Journal Article %J Microb Biotechnol %D 2018 %T Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. %A Bairy, Sneha %A Gopalan, Lakshmi Narayanan %A Setty, Thanuja Gangi %A Srinivasachari, Sathya %A Manjunath, Lavanyaa %A Kumar, Jay Prakash %A Guntupalli, Sai R %A Bose, Sucharita %A Nayak, Vinod %A Ghosh, Swagatha %A Sathyanarayanan, Nitish %A Caing-Carlsson, Rhawnie %A Wahlgren, Weixiao Yuan %A Friemann, Rosmarie %A Ramaswamy, S %A Neerathilingam, Muniasamy %X

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

%B Microb Biotechnol %V 11 %P 420-428 %8 2018 Mar %G eng %N 2 %R 10.1111/1751-7915.13041 %0 Journal Article %J Acta Crystallogr F Struct Biol Commun %D 2017 %T Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. %A Caing-Carlsson, Rhawnie %A Goyal, Parveen %A Sharma, Amit %A Ghosh, Swagatha %A Setty, Thanuja Gangi %A North, Rachel A %A Friemann, Rosmarie %A Ramaswamy, S %K Adenosine Triphosphate %K Amino Acid Sequence %K Bacterial Proteins %K Binding Sites %K Cloning, Molecular %K Crystallography, X-Ray %K Escherichia coli %K Fusobacterium nucleatum %K Gene Expression %K Genetic Vectors %K Hexosamines %K Models, Molecular %K Phosphotransferases (Alcohol Group Acceptor) %K Protein Binding %K Protein Conformation, alpha-Helical %K Protein Conformation, beta-Strand %K Protein Interaction Domains and Motifs %K Protein Multimerization %K Recombinant Proteins %K Sequence Alignment %K Sequence Homology, Amino Acid %K Substrate Specificity %X

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

%B Acta Crystallogr F Struct Biol Commun %V 73 %P 356-362 %8 2017 Jun 01 %G eng %N Pt 6 %R 10.1107/S2053230X17007439 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2016 %T Blue protein with red fluorescence. %A Ghosh, Swagatha %A Yu, Chi-Li %A Ferraro, Daniel J %A Sudha, Sai %A Pal, Samir Kumar %A Schaefer, Wayne F %A Gibson, David T %A Ramaswamy, S %K Biliverdine %K Crystallography, X-Ray %K Fluorescence %K Models, Molecular %K Proteins %K Recombinant Proteins %X

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λ of 375 nm) and upon excitation at this wavelength emits in the red region (λ of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

%B Proc Natl Acad Sci U S A %V 113 %P 11513-11518 %8 2016 10 11 %G eng %N 41 %R 10.1073/pnas.1525622113