期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 291, 期 8, 页码 4226-4235出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M115.673871
关键词
atomic force microscopy (AFM); cataract; crystallin; protein misfolding; single-molecule biophysics
资金
- Marie Curie Intra-European Fellowships for Career Development Grant [329308]
- Marie Curie Carrier Integration Grants [293462]
- Biotechnology and Biological Sciences Research Council [BB/J00992X/1]
- Engineering and Physical Sciences Research Council [K00641X/1]
- Royal Society [RG120038]
- National Institutes of Health [HL66030, HL61228]
- BBSRC [BB/J00992X/1] Funding Source: UKRI
- EPSRC [EP/K00641X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/J00992X/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/K00641X/1, 1406913] Funding Source: researchfish
Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human D-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HD-crystallin monomers, we captured an anomalous misfolded conformation in which the 1 and 2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.
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