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Deamidation of Asn residues, which results in formation of a mixture of <jats:sc>l<\/jats:sc>\u2010 and <jats:sc>d<\/jats:sc>\u2010Asp and <jats:italic>iso<\/jats:italic>Asp via an intermediate aspartyl succinimide, can result in the disruption of cellular proteostasis and toxic protein depositions. In contrast to extensive data on the biological prevalence and functional implications of aspartyl succinimide formation, much less is known about the impact of the resulting altered backbone composition on properties of individual proteins at a molecular level. Here, we report the total chemical synthesis, biophysical characterization, and NMR structural analysis of a series of variants of the B1 domain of protein G from <jats:italic>Streptococcal<\/jats:italic> bacteria (GB1) in which all possible Asp isomers as well as an aspartyl succinimide were individually incorporated at a defined position in a solvent\u2010exposed loop. Subtle local structural effects were observed; however, these were accompanied by notable differences in thermodynamic folded stability. Surprisingly, the noncanonical backbone connectivity of <jats:sc>d<\/jats:sc>\u2010<jats:italic>iso<\/jats:italic>Asp led to a variant that exhibited enhanced stability relative to the natural protein.<\/jats:p>","DOI":"10.1002\/pro.4883","type":"journal-article","created":{"date-parts":[[2023,12,25]],"date-time":"2023-12-25T08:38:45Z","timestamp":1703493525000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Probing effects of site\u2010specific aspartic acid isomerization on structure and stability of <scp>GB1<\/scp> through chemical protein synthesis"],"prefix":"10.1002","volume":"33","author":[{"given":"Shelby L.","family":"Heath","sequence":"first","affiliation":[{"name":"Department of Chemistry University of Pittsburgh  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex J.","family":"Guseman","sequence":"additional","affiliation":[{"name":"Department of Structural Biology University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9072-3525","authenticated-orcid":false,"given":"Angela M.","family":"Gronenborn","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Pittsburgh  Pittsburgh Pennsylvania USA"},{"name":"Department of Structural Biology University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2927-1739","authenticated-orcid":false,"given":"W. 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