Dissecting the Functional Domains of Phi11 Gp13 through Deletion Mutagenesis.

Dissecting the Functional Domains of Phi11 Gp13 through Deletion Mutagenesis. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 31 October 2025 V1 Latest version Share on Dissecting the Functional Domains of Phi11 Gp13 through Deletion Mutagenesis. Authors : Vivek Ratre and Malabika Biswas 0000-0002-2497-9765 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176187795.59817508/v1 168 views 124 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Single-stranded DNA-binding proteins (SSBs) are found across all life domains, including viruses. They stabilize transient single-stranded DNA (ssDNA) intermediates, vital for nucleic acid metabolism. While bacterial SSBs typically form homotetramers with an N-terminal oligonucleotide/oligosaccharide binding (OB) fold, the bacteriophage Phi11 Gp13 is a homodimer with a C-terminal OB-fold. This study investigates functional domains in Phi11 Gp13 via biochemical and biophysical characterization of N and C-terminal deletion mutants. Specific N-terminal (residues 2-8) and C-terminal (residues 177-184) residues are critical for efficient ssDNA binding affinity. Notably, C-terminal (C1, C2) and N-terminal (N1) deletions resulted in a novel shift in binding site size from 25 nucleotides (for Gp13) to 20 nucleotides, suggesting peripheral residues impose structural constraints on the DNA-binding domain. On the contrary, further N-terminal deletions, as seen in case of N5, completely abolished DNA binding, highlighting the importance of residues R6-E12 in forming the DNA-binding surface. Despite exhibiting differential ssDNA binding, most mutants retained their dimeric state, suggesting the dimerization domain lies outside the terminal regions of the protein. While overall structural integrity was largely preserved, localized secondary and tertiary changes (e.g., increased alpha-helical content, subtle red shifts in tryptophan fluorescence, destabilizations in C2 and N5) were observed, which correlated directly with ssDNA binding affinity. Furthermore, Gp13 interacted with Staphylococcus aureus ( S. aureus ) RecA. This research reveals the important links between the primary amino acid sequence, its three-dimensional conformation, and the biological function of Gp13. Supplementary Material File (figures _proteins.docx) Download 2.04 MB File (manuscript proteins updated.docx) Download 152.25 KB File (tables _proteins.docx) Download 33.67 KB Information & Authors Information Version history V1 Version 1 31 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords acidic tail dna binding emsa ob-fold protein interaction pull-down assay pxxp motif Authors Affiliations Vivek Ratre Birla Institute of Technology & Science Pilani - K K Birla Goa Campus View all articles by this author Malabika Biswas 0000-0002-2497-9765 [email protected] Birla Institute of Technology & Science Pilani - K K Birla Goa Campus View all articles by this author Metrics & Citations Metrics Article Usage 168 views 124 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Vivek Ratre, Malabika Biswas. Dissecting the Functional Domains of Phi11 Gp13 through Deletion Mutagenesis.. Authorea . 31 October 2025. DOI: https://doi.org/10.22541/au.176187795.59817508/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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