Examination of Aggregation Prone Proteins and their Higher Order Interactions by Ion Mobility-Mass Spectrometry.

Protein Aggregration;Amyloid Beta;Ion Mobility Mass Spectrometry;Biological Chemistry;Chemistry;Health Sciences;Science;Chemistry

Soper, Molly ThereseRamamoorthy, Ayyalusamy ;

University of Michigan

关键词: Protein Aggregration;    Amyloid Beta;    Ion Mobility Mass Spectrometry;    Biological Chemistry;    Chemistry;    Health Sciences;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/116673/mtso_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Amyloid forming peptides and proteins present an extreme challenge for modern analytical measurement techniques. When natively-folded biomolecules partition into misfolded forms, they produce a myriad of inherently unstable conformations that eventually lead to the presumed cytotoxic species linked to disease. Most often, these cytotoxic states take the form of small oligomeric species that exist within a complex mixture of other oligomers that may, or may not, be related to a disease etiology. In addition, these dynamically-generated oligomers are present in small amounts, within a complex mixture of other biomolecules, small molecules, and metal ions that may also influence the ensemble of states being measured. Ion mobility separation, coupled to mass spectrometry, has recently become a key technology for the analysis of amyloid-forming peptides and proteins due to its ability to analyze low concentrations of complex mixtures. Described here is the application of IM-MS to study protein interactions with small molecules, dipeptides, and neuropeptides. This combine information furthers the knowledge base of neurodegenerative diseases from a structural standpoint.First, the interaction of amyloid beta: leucine enkephalin was discovered in a screen of many neuropeptides. This interaction was characterized using IM-MS through collision cross section and Kd measurements. Multiple copies of leucine enkephalin are found to bind with amyloid beta, each with an approximate binding strength of 60 micromolar. Modeling suggests binding near the structured core region of amyloid beta. Following these initial studies, site directed amino acid substitution of leucine enkephalin reveals that the hydrophobic C-terminal residues phenylalanine and leucine are critical for binding to occur. Alanine substitutions of amyloid beta residues, selected based on simulated annealing results, indicate residues Y10 and Q15 to be most critical for the interaction of amyloid beta with the dipeptide FL. Molecular dynamics models were filtered using IM-MS experimental results, and structures representative of the interaction are shown. Additionally, workflow to study amyloid beta has been applied to the oligomerization of ubiquitin monomer and linear tri-ubiqutin in the presence of copper ions, measuring an increase in the number of dimeric species. Stability measurements using collision induced unfolding, and aggregation propensity of Ubiquilin2 are also studied.