Abstract
The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution into the structural changes of profilin-1 (PFN-1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN-1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN-1 protein dynamic behavior using purified wild type and mutant PFN-1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN-1WT and PFN-1G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN-1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN-1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFNG117V. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN-1s' dimerization, oligomerization, and aggregation.
Highlights
Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder that characterized by the degeneration of both upper and lower motor neurons (Brown and Al-Chalabi 2017, Hardiman et al 2017)
We sought to investigate the structural changes in mutant PFN-1 using in silico tools combined with hydrogen-deuterium exchange linked with mass spectrometry (HDX-Mass spectrometry (MS)) techniques (Pirrone et al 2017)
A small volume from each fraction was prepared to load on SDS-Polyacrylamide gel electrophoresis (PAGE) to analyze the expressed proteins and confirmed the protein in the band around size (14–15 kDa) as PFN-1, as it is close to the size of the recombinant PFN-1 (Fig. 3S)
Summary
Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder that characterized by the degeneration of both upper and lower motor neurons (Brown and Al-Chalabi 2017, Hardiman et al 2017). The average age of survival for ALS patients is 2–3 years onset of symptoms (Millul et al 2005). There are two FDA-approved drugs exist for ALS (Riluzole and Radicava). These drugs have marginal benefits and don’t stop the symptoms of ALS. In order to develop a novel and effective therapy, understanding the mechanism(s) of toxicities caused by mutant proteins or other factors is essential. One of the mutant proteins linked to ALS is profilin-1 (PFN-1) (Wu et al 2012). The exact mechanism and the specific of mutant profilin-1 toxicities aren’t fully understood. Understanding the mechanism of PFN-1 toxicity could be a key in unravelling how mutant proteins play a role in the degeneration of motor neurons. We sought to investigate the structural changes in mutant PFN-1 using in silico tools combined with hydrogen-deuterium exchange linked with mass spectrometry (HDX-MS) techniques (Pirrone et al 2017)
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