Structural and sequence analysis of the human γD-crystallin amyloid fibril core using 2D IR spectroscopy, segmental 13C labeling, and mass spectrometry.
Identifying the sequence and structural content of residues that compose the core of amyloid fibrils is important because core regions likely control the process of fibril extension and provide potential drug targets. Human γD-crystallin is an eye lens protein that aggregates into amyloid fibrils under acidic conditions. In this manuscript, we use a pepsin enzymatic digest to isolate the core of the amyloid fibrils. The sequence of the core is identified with MALDI MS/MS and its structure is probed with 2D IR spectroscopy and (13)C isotope labeling. Mass spectrometry of the digest identifies residues 80-163 as the amyloid core, which spans most of the C-terminal domain, the linker, and a small portion of the N-terminal domain. From 2D IR spectroscopy of the digested fibrils, we learn that only the C-terminal domain contributes to the amyloid β-sheets while the N-terminal and linker residues are disordered. A comparison to the native crystal structure reveals that loops and α-helices in the native state must undergo conformational transitions to β-strands upon aggregation. These locations may be good drug binding targets. Besides providing new information about γD-crystallin, this study demonstrates the complementarity of mass spectrometry and 2D IR spectroscopy to obtain both sequence and structure information that neither technique provides individually, which will be especially useful for samples only available in microgram quantities.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
Sean D
SD
Moran
Sean M
SM
Decatur
Martin T
MT
Zanni
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D002247:Carbon Isotopes / Q000032:analysis; D006801:Humans; D007553:Isotope Labeling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013055:Spectrophotometry, Infrared; D053719:Tandem Mass Spectrometry; D038222:gamma-Crystallins / Q000737:chemistry*
D000682:Amyloid; C504903:CRYGD protein, human; D002247:Carbon Isotopes; D038222:gamma-Crystallins