Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry

Abstract

Actin-related protein (Arp) 2/3 complex nucleates new branches in actin filaments playing a key role in controlling eukaryotic cell motility. This process is tightly regulated by activating factors: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We used radiolytic protein footprinting with mass spectrometry in solution to probe the effects of nucleotide-and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting approaches.

A Hybrid Structural Approach to Analyze Ligand Binding by the Serotonin Type 4 Receptor (5-HT4)

“…Calculation of Oxidation Rates-The fraction of unoxidized peptides was calculated from the ratio of the chromatographic area under the ion signals for the unoxidized peptides to the sum of the unoxidized peptides and their radiolytic products (22,24). Dose-response curves are presented as unmodified fractions versus exposure peri-…”

“…S6). The fraction of unoxidized peptides was calculated from the ratio of the chromatographic area under the ion signals for the unoxidized peptides to the sum of the unoxidized peptides and their radiolytic products (22,24 4.62 , which is located on C terminus of TM4) were found to be modified and spatially distributed on the extracellular side of the receptor in addition to E11 of the N terminus (Fig. 1).…”

Structural waters define a functional channel mediating activation of the GPCR, rhodopsin

“…This indicates that radicals that modify residues within the helical bundle are formed in situ in intramembranous regions of rhodopsin, photoactivated receptor, and apo-protein. Only following dehydration and rehydration of rhodopsin with 97% H 2O 18 water and subsequent X-ray exposure were low levels of O 18 pocket and increased labeling of side chains in these helices following receptor activation.…”

“…In proteins, the chemistry of modification is well-established, and many types of side chain can be efficiently labeled and the products easily detected by mass spectrometry, although the dynamic range of labeling varies considerably, with aromatic and sulfur containing side chains easily labeled and aliphatic and polar residues labeled less efficiently (15,16). For soluble proteins, it is well-established that this intrinsic reactivity and the solvent accessibility of the side chains govern their observed reactivity (15,17,18). For membrane proteins, these approaches have not been previously explored, such that it was unclear what factors would govern labeling, or whether the overall scavenging effects of detergents or lipids are limiting.…”

Fragmentation mechanisms of oxidized peptides elucidated by SID, RRKM modeling, and molecular dynamics

“…Elucidation of the collisional fragmentation patterns of oxidized peptides also pertains to a protein characterization method referred to as oxidative footprinting [35][36][37][38][39][40][41][42][43][44][45][46][47]. In this technique, three-dimensional structures and conformational changes of proteins and protein complexes are examined by observing the oxidation reactivity of the solvent accessible amino acid side chains.…”