Determination of absolute intramolecular distances in proteins using anomalous X-ray scattering interferometry

Samuel Stubhan, Anna V. Baptist, Caroline Körösy, Alessandra Narducci, Gustavo Gabriel Moya Muñoz, Nicolas Wendler, Aidin Lak, Michael Sztucki, Thorben Cordes*, Jan Lipfert*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance or distance distributions. Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.

Original languageEnglish
JournalNanoscale
DOIs
Publication statusE-pub ahead of print - 9 Dec 2024

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© 2025 The Royal Society of Chemistry.

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