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Ligand-induced conformational changes in protein molecules detected by sum-frequency generation (SFG).
Salafsky, Joshua; Johansson, Patrik K; Abdelkader, Elwy; Otting, Gottfried.
Afiliación
  • Salafsky J; Department of Pharmaceutical Chemistry, University of California, San Francisco (UCSF), Genentech Hall Box 2280, 600 16(th) St., Rm 518, San Francisco CA 94158; Skylight Discovery, Inc. Suite 300, 4500 9(th) Avenue NE, Seattle WA 98115. Electronic address: joshua.salafsky@ucsf.edu.
  • Johansson PK; Skylight Discovery, Inc. Suite 300, 4500 9(th) Avenue NE, Seattle WA 98115.
  • Abdelkader E; ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Otting G; ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
Biophys J ; 2024 Sep 19.
Article en En | MEDLINE | ID: mdl-39305014
ABSTRACT
We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRasG12D protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis (CFPS). By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein's native structure. Exceptionally large SFG amide I signals were produced in both labeled and unlabeled proteins, demonstrating a high degree of orientational order upon attachment to the bilayer. Deuterated protein also produced SFG signals in the CDx spectral region, which were not present in the unlabeled protein. The CDx signals were measured before and after binding a peptide inhibitor, KRpep-2d, revealing shifts in SFG intensity due to conformational changes at the labeled sites. In particular, peaks associated with CDx stretching vibrations for alanine, valine, and glycine changed substantially in amplitude upon inhibitor binding. By inspection of the crystal structure, these three residues are uniquely co-located on the protein surface in and near the nucleotide binding site, which is in allosteric communication with the site of peptide inhibitor binding, suggesting an approach to identify a ligand's binding site. The technique offers a highly sensitive, non-perturbative method of mapping ligand-induced conformational changes and allosteric networks in biological molecules for studies of the relationship between structure and function and mechanisms of action in drug discovery.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Biophys J Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Biophys J Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos