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  • 标题:Low-energy electron holography imaging of conformational variability of single-antibody molecules from electrospray ion beam deposition
  • 本地全文:下载
  • 作者:Hannah Ochner ; Sven Szilagyi ; Sabine Abb
  • 期刊名称:Proceedings of the National Academy of Sciences
  • 印刷版ISSN:0027-8424
  • 电子版ISSN:1091-6490
  • 出版年度:2021
  • 卷号:118
  • 期号:51
  • DOI:10.1073/pnas.2112651118
  • 语种:English
  • 出版社:The National Academy of Sciences of the United States of America
  • 摘要:Significance Molecular imaging at the single-molecule level of large and flexible proteins such as monoclonal IgG antibodies is possible by low-energy electron holography after chemically selective sample preparation by native electrospray ion beam deposition (ES-IBD) from native solution conditions. The single-molecule nature of the measurement allows the mapping of the structural variability of the molecules that originates from their intrinsic flexibility and from different adsorption geometries. Additionally, we can distinguish gas-phase–related conformations and conformations induced by the landing of the molecules on the surface. Our results underpin the relation between the gas-phase structure of protein ions created by native electrospray ionization (ESI) and the native protein structure and are of relevance for structural biology applications in the gas phase. Imaging of proteins at the single-molecule level can reveal conformational variability, which is essential for the understanding of biomolecules. To this end, a biologically relevant state of the sample must be retained during both sample preparation and imaging. Native electrospray ionization (ESI) can transfer even the largest protein complexes into the gas phase while preserving their stoichiometry and overall shape. High-resolution imaging of protein structures following native ESI is thus of fundamental interest for establishing the relation between gas phase and solution structure. Taking advantage of low-energy electron holography’s (LEEH) unique capability of imaging individual proteins with subnanometer resolution, we investigate the conformational flexibility of Herceptin, a monoclonal IgG antibody, deposited by native electrospray mass-selected ion beam deposition (ES-IBD) on graphene. Images reconstructed from holograms reveal a large variety of conformers. Some of these conformations can be mapped to the crystallographic structure of IgG, while others suggest that a compact, gas-phase–related conformation, adopted by the molecules during ES-IBD, is retained. We can steer the ratio of those two types of conformations by changing the landing energy of the protein on the single-layer graphene surface. Overall, we show that LEEH can elucidate the conformational heterogeneity of inherently flexible proteins, exemplified here by IgG antibodies, and thereby distinguish gas-phase collapse from rearrangement on surfaces.
  • 关键词:low-energy electron holography; single-molecule imaging; native electrospray ion beam deposition
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