{"paper_id":"1af89b6b-e140-44de-8ffd-4d0fdf6d1999","body_text":"Evolutionary and Physics-Guided Modulation of Ca2+ Sensitivity in Cardiac Troponin C | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evolutionary and Physics-Guided Modulation of Ca 2+ Sensitivity in Cardiac Troponin C Abdul Basit This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8734817/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Calcium sensitivity in cardiac muscle must be precisely tuned to enhance systolic force generation without impairing diastolic relaxation. Here we present a physics- and evolution-guided strategy to achieve modest, physiologically relevant Ca 2+ sensitization of cardiac troponin C by targeting its sole regulatory EF-hand. Using Potts-model-based evolutionary constraints as a form of negative design, we identify mutations that preserve native allosteric architecture while excluding perturbations that artificially stabilize the Ca 2+ -bound state. Molecular dynamics simulations show that effective sensitization does not arise from increased Ca 2+ affinity or nonspecific loop softening, but from enhanced coupling between Ca 2+ motion and EF-hand conformational response in the bound-state. These results establish a physical mechanism for tunable Ca 2+ regulation and demonstrate how evolutionary constraints can be exploited to engineer regulatory proteins without compromising physiological control. Biophysics Protein engineering calcium signaling Cardiac troponin C allosteric regulation evolutionary constraints Full Text Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8734817\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":582659478,\"identity\":\"f2deb98c-ef25-4f36-b421-9571628794b0\",\"order_by\":0,\"name\":\"Abdul Basit\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYLACxgYGBjYG5gNApoQMcRrAWtjYEkBaeIjXwsDGYwDiEdai2958/MHPHXb2fPI9n1/dqLHgYWA/fHQDPi1mZ44lNvaeSU5sY+PdZp1zDOgwnrS0G3i13MgxbOBtY05gA2oxzmEDapHgMcOv5f77j41/2+rt2dh4nhnn/CNGyw0exmbetsOMbWw8zI9z24jRcibNcLbsmeNAv6SZMef2SfCwEfTL8cMPPr7dUW0v33z48eecb3Vy/OyHj+HVggzYJMAkscpBgPkDKapHwSgYBaNg5AAAmVtGgn6hUJsAAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0001-7473-8401\",\"institution\":\"Jawaharlal Nehru University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Abdul\",\"middleName\":\"\",\"lastName\":\"Basit\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-01-29 19:44:45\",\"currentVersionCode\":1,\"declarations\":{\"humanSubjects\":false,\"vertebrateSubjects\":false,\"conflictsOfInterestStatement\":false,\"humanSubjectEthicalGuidelines\":false,\"humanSubjectConsent\":false,\"humanSubjectClinicalTrial\":false,\"humanSubjectCaseReport\":false,\"vertebrateSubjectEthicalGuidelines\":false},\"doi\":\"10.21203/rs.3.rs-8734817/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8734817/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":101754458,\"identity\":\"42e02ea6-2a01-4076-a7e3-88828b0cddbd\",\"added_by\":\"auto\",\"created_at\":\"2026-02-03 10:42:32\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1099931,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ms.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8734817/v1_covered_0547a7bb-7f2f-4755-8455-9a2baac4dff6.pdf\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"\\u003cp\\u003eEvolutionary and Physics-Guided Modulation of Ca\\u003csup\\u003e2+\\u003c/sup\\u003e Sensitivity in Cardiac Troponin C\\u003c/p\\u003e\",\"fulltext\":[],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":false,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"Jawaharlal Nehru University\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":true,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":true,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Protein engineering; calcium signaling; Cardiac troponin C; allosteric regulation; evolutionary constraints;\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8734817/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8734817/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eCalcium sensitivity in cardiac muscle must be precisely tuned to enhance systolic force generation without impairing diastolic relaxation. 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