A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes

Abstract

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring‐shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light‐harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.

Predicting the Shapes of Protein Complexes through Collision Cross Section Measurements and Database Searches

“…The PDB mining was carried out as described previously . Briefly, masses and helium CCS for the SAP pentamer and decamer, and the bovine lactoglobulin dimer were taken from the Bush CCS database or determined experimentally by TWIMS for NHA2 and NapA (see below).…”

“…There is a trove of general structural information about proteins, because protein structures are not random: besides the selection that has given rise to specific functions, they have all evolved under common biophysical constraints that dictate what sizes, shapes, and architectures are beneficial. − It is likely that this has shaped the structural proteome on many levels, creating patterns in how the structural space is populated, which in turn could be modulated by other properties, such as the oligomeric state or subcellular location. These patterns may in part be revealed by inspecting collections of known protein structures, and structural databases linking high-resolution structures and CCS information can consequently be used to indicate the architectures of protein complexes with unknown structures …”

“…These patterns may in part be revealed by inspecting collections of known protein structures, and structural databases linking high-resolution structures and CCS information can consequently be used to indicate the architectures of protein complexes with unknown structures. 14 Here, by mining the PDB using only molecular weight, CCS, and oligomeric state information, we have determined that it is possible to predict whether a protein complex adopts an oblate or a prolate shape, or a spherical architecture. This simple classification of protein shape can reduce the search space for low-resolution models without a need for reference structures or complex computations.…”

The Ims Paradox: A Perspective on Structural Ion Mobility‐mass Spectrometry

“…Moreover, despite several studies attempting to correlate the ion mobility measurements to exact shape of ions (Kaldmaee et al, 2019; Landreh et al, 2020), a priori assignment of detailed structure on the basis of rotationally averaged collision cross section (CCS) is not possible; therefore, it is common practice to compare the measured CCS to calculated CCS for structures obtained from NMR, circular dichroism (CD), crystallography, and/or MD simulations.…”

Predicting the Shapes of Protein Complexes Through Collision Cross Section Measurements and Database Searches

“…15 By mining the PDB for protein complexes that match an experimentally determined CCS, we were, for example, able to confirm the ring-like shape of a phycobiliprotein complex with an additional subunit. 14 These findings led us to consider the PDB as a large collection of sample structures, with the additional advantage that the structures represent predominantly physiologically relevant assemblies. Therefore, by mining the PDB for protein complexes with similar stoichiometry, MW, and CCS, it may be possible to identify which shape(s) an unknown protein complex is likely to adopt.…”