{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T14:28:58Z","timestamp":1786631338325,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,11]]},"group-title":"Biochemistry","reference-count":40,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:00:00Z","timestamp":1786406400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"funder":[{"award":["R01GM147635"],"award-info":[{"award-number":["R01GM147635"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P20GM103418"],"award-info":[{"award-number":["P20GM103418"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T34GM136453"],"award-info":[{"award-number":["T34GM136453"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>A central challenge in molecular biology is understanding how amino acid substitutions modulate various features of protein function and stability. To illuminate the complexities of this relationship, high-throughput (HTP) assays are increasingly used to assess site-saturating mutagenesis libraries. A common downstream analysis is to average the set of twenty outcomes at each amino acid position for comparison with structural and evolutionary features. Average values clearly identify positions that tolerate most substitutions (neutral positions) and positions where most substitutions abolish activity (toggle positions). However, average values conceal the existence of rheostat positions, where different amino acid substitutions sample a wide range of outcomes. To quantitatively identify rheostat positions, we previously developed a histogram-based analysis that we here expand by: (i) incorporating new position classes observed in experimental studies of rheostat positions; (ii) formalizing a hierarchy of class assignments; (iii) refining error-based identification of neutral positions; and (iv) statistically assessing the robustness of class assignments to changes in experimental and computational parameters. RheoScale 2.0 is implemented in Excel and newly implemented in Python for facile integration with existing HTP pipelines; all parameters are customizable. Example analyses are shown for three HTP datasets of the SARS-CoV-2 papain-like protease. Results illustrate two aspects that influence interpretation of HTP data: First, position assignments (and substitution outcomes) depend highly on the measured feature. Second, many protein positions play multiple roles in the sequence-structure-function relationship. The recognition of varied position roles will advance understanding of pathogen evolution, protein engineering, and variant interpretation for personalized medicine.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Summary<\/jats:title>\n                  <jats:p>RheoScale 2.0 improves how high-throughput mutational data are interpreted by identifying protein positions where amino acid substitutions act like biological dimmer switches. By enabling more nuanced assignment of position behavior, beyond neutral or deleterious outcomes, this analysis framework advances studies of sequence-structure-function relationships and has broad relevance for understanding protein evolution, engineering proteins with desired properties, and interpreting variants linked to human disease.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>SOFTWARE AVAILABILITY<\/jats:title>\n                  <jats:p>\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/liskinsk\/RheoScale-calculator\">https:\/\/github.com\/liskinsk\/RheoScale-calculator<\/jats:ext-link>\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.10.743964","type":"posted-content","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:50:20Z","timestamp":1786510220000},"source":"Crossref","is-referenced-by-count":0,"title":["RheoScale 2.0: Revealing the Hidden Roles of Protein Positions via Substitution Patterns"],"prefix":"10.64898","author":[{"given":"David H.","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA 66106;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5869-4254","authenticated-orcid":false,"given":"Shwetha","family":"Sreenivasan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA 66106;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carter J.","family":"Gray","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA 66106;"},{"name":"Department of Electrical Engineering and Computer Science, University of Kansas-Lawrence, Lawrence, KS, 66045"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah C.","family":"Cleveland","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA 66106;"},{"name":"Department of Electrical Engineering and Computer Science, University of Kansas-Lawrence, Lawrence, KS, 66045"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5925-9741","authenticated-orcid":false,"given":"Liskin","family":"Swint-Kruse","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA 66106;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081306350927000_2026.08.10.743964v1.1","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1007\/s00439-024-02724-8","article-title":"Assessing the predicted impact of single amino acid substitutions in MAPK proteins for CAGI6 challenges","volume":"144","year":"2025","journal-title":"Hum Genet"},{"key":"2026081306350927000_2026.08.10.743964v1.2","doi-asserted-by":"crossref","first-page":"lqae154","DOI":"10.1093\/nargab\/lqae154","article-title":"There will always be variants of uncertain significance","volume":"6","year":"2024","journal-title":"Analysis of VUSs. 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