Conformational changes of recombinant monoclonal antibodies by limited proteolytic digestion, stable isotope labeling, and liquid chromatography–mass spectrometry

Ponniah, Gomathinayagam; Nowak, Christine; Kita, Adriana; Cheng, Guilong; Kori, Yekaterina; Liu, Hongcheng

doi:10.1016/j.ab.2015.12.008

Cited by 2 publications

(1 citation statement)

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“…Figure 3 shows that not only lysines but also arginines are located on both sides of the mTSPO. Both types of residues are selectively digested by trypsin; thus, we took advantage of this to perform a limited proteolysis digestion that has been previously used to probe the conformational changes of proteins [20,21].…”

Section: Resultsmentioning

confidence: 99%

Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

Iatmanen-Harbi

Senicourt

Papadopoulos

et al. 2019

IJMS

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The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

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Section: Resultsmentioning

confidence: 99%