Show simple item record

dc.contributor.authorLobner, Elisabeth
dc.contributor.authorTraxlmayr, Michael
dc.contributor.authorKiefer, Jonathan
dc.contributor.authorSrinivas, Raja Ram
dc.contributor.authorTisdale, Alison W.
dc.contributor.authorMehta, Naveen
dc.contributor.authorYang, Nicole Jie Yeon
dc.contributor.authorTidor, Bruce
dc.contributor.authorWittrup, Karl Dane
dc.date.accessioned2017-06-13T17:35:49Z
dc.date.available2017-06-13T17:35:49Z
dc.date.issued2016-08
dc.date.submitted2016-08
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.urihttp://hdl.handle.net/1721.1/109823
dc.description.abstractThe Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.en_US
dc.description.sponsorshipUnited States. National Institutes of Health (CA174795)en_US
dc.description.sponsorshipUnited States. National Institutes of Health (CA96504)en_US
dc.language.isoen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology (ASBMB)en_US
dc.relation.isversionofhttp://dx.doi.org/10.1074/jbc.M116.741314en_US
dc.rightsCreative Commons Attribution 4.0 International Licenseen_US
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en_US
dc.sourceAmerican Society for Biochemistry and Molecular Biologyen_US
dc.titleStrong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Libraryen_US
dc.typeArticleen_US
dc.identifier.citationTraxlmayr, Michael W.; Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce and Wittrup, K. Dane. “Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-Neutralized Hyperthermostable Sso7d Scaffold Library.” Journal of Biological Chemistry 291, no. 43 (August 2016): 22496–22508 © 2016 American Society for Biochemistry and Molecular Biology (ASBMB)en_US
dc.contributor.departmentDavid H. Koch Institute for Integrative Cancer Research at MITen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biological Engineeringen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemical Engineeringen_US
dc.contributor.mitauthorTraxlmayr, Michael
dc.contributor.mitauthorKiefer, Jonathan
dc.contributor.mitauthorSrinivas, Raja Ram
dc.contributor.mitauthorTisdale, Alison W.
dc.contributor.mitauthorMehta, Naveen
dc.contributor.mitauthorYang, Nicole Jie Yeon
dc.contributor.mitauthorTidor, Bruce
dc.contributor.mitauthorWittrup, Karl Dane
dc.relation.journalJournal of Biological Chemistryen_US
dc.eprint.versionFinal published versionen_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsTraxlmayr, Michael W.; Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Daneen_US
dspace.embargo.termsNen_US
dc.identifier.orcidhttps://orcid.org/0000-0002-2108-582X
dc.identifier.orcidhttps://orcid.org/0000-0002-5476-903X
dc.identifier.orcidhttps://orcid.org/0000-0002-3161-2785
dc.identifier.orcidhttps://orcid.org/0000-0003-3480-6750
dc.identifier.orcidhttps://orcid.org/0000-0002-0882-7761
dc.identifier.orcidhttps://orcid.org/0000-0002-3320-3969
dc.identifier.orcidhttps://orcid.org/0000-0003-2398-5896
mit.licensePUBLISHER_CCen_US


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record