Detection of structural and conformational changes in ALS-causing mutant profilin-1 with hydrogen/deuterium exchange mass spectrometry and bioinformatics techniques

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 in the structural changes of profilin1 (PFN1). 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 PFN1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN1 protein dynamic behavior using purified wild type and mutant PFN1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN1 WT and PFN1 G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFN G117V . This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN1s' dimerization, oligomerization, and aggregation.

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

“…Wild-type profilin has efficient folding dynamics and can refold following several denaturization procedures (Kaiser et al, 1989). Several studies have explicitly detailed folding and stability defects with certain ALS-linked profilin variants (Boopathy et al, 2015; Del Poggetto et al, 2016; Freischmidt et al, 2015; Kiaei et al, 2018; Pereira et al, 2019; Sadr et al, 2021). The solubility and stability of each profilin variant is an important consideration for this study.…”

“…Several studies suggest that at least a subset of these ALS-linked profilin proteins mildly destabilize actin dynamics in cells (Boopathy et al, 2015; Figley et al, 2014; Freischmidt et al, 2015; Schmidt et al, 2021; Wu et al, 2012). Other analyses imply that C71G, T109M, M114T, G118V, and to a lesser extent E117G misfold in ALS-pathology (Boopathy et al, 2015; Del Poggetto et al, 2016; Figley et al, 2014; Kiaei et al, 2018; Pereira et al, 2019; Sadr et al, 2021). Whether the ALS-related profilin variants directly disrupt actin binding or formin-based assembly mechanisms has not been fully explored.…”

Rapid antibody conformational screening by matrix‐assisted laser desorption/ionization hydrogen‐deuterium exchange mass spectrometry

“…The problem of back exchange was avoided by using a MALDI matrix that was dissolved completely in a non-aqueous solvent system [13]. Since then, several studies have been dedicated to MALDI-HDX measurements, with protein conformation, dynamics, and stability being the topics of interest [12,25,26]. The goal of this study was to demonstrate the ability of MALDI-HDX-MS to unambiguously detect conformational differences between intact BsAbs from different expression hosts in a rapid and high-throughput manner.…”

Correlation between Ki-67 or Profilin-1 Expression Levels, Clinicopathological Characteristics and Postoperative Prognosis in Patients with Bladder Cancer

“…Its expression level is closely related to clinical pathological parameters, which can reflect the malignancy of the tumor from the side, and plays an auxiliary observation role in the diagnosis and prognosis of bladder cancer [9]. Profilin-1 (PFN1) is an actin-binding protein and one of the key proteins involved in fiber actin dynamics [10]. PFN1 interacts with various proteins and participates in pivotal biological processes, including cell proliferation, cell survival, and cell division [11].…”