title: Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.
abstract: Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.
authors.affiliation: From the ‡Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom;
authors.forename: Adam
authors.initials: A
authors.lastname: Belsom
authors.affiliation: §Robotics and Biology Laboratory, Technische Universität Berlin, 10587 Berlin, Germany;
authors.forename: Michael
authors.initials: M
authors.lastname: Schneider
authors.affiliation: From the ‡Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom;
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
authors.affiliation: §Robotics and Biology Laboratory, Technische Universität Berlin, 10587 Berlin, Germany;
authors.forename: Oliver
authors.initials: O
authors.lastname: Brock
authors.affiliation: From the ‡Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom; ¶Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany. juri.rappsilber@ed.ac.uk.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
mesh_terms: D019295:Computational Biology / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D055668:Photochemical Processes; D000072417:Protein Domains; D012709:Serum Albumin / Q000737:chemistry* / Q000302:isolation & purification; D000075462:Serum Albumin, Human; D013388:Succinimides / Q000737:chemistry; D014631:Valerates / Q000737:chemistry
chemical_list: C422455:ALB protein, human; D003432:Cross-Linking Reagents; D012709:Serum Albumin; D013388:Succinimides; D014631:Valerates; D000075462:Serum Albumin, Human