Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2

Manandhar, Pragya; Mazhar, Zahra; Abousaway, Omar; Aboagye, Collins; Moussa, Zeinab; Lim, Daniel V.; Yu, Tannon; Byrnes, James R.; Briggs, James M.; Şen, Mehmet

doi:10.1016/j.celrep.2022.111254

Cited by 1 publication

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“…DSF assays were used to characterize the thermal stability of proteins and of complexes of linear and cyclic Z3 with IgG 1 [ 55 ]. Proteins (32 μM) were mixed with 10X SYPRO™ orange dye (stock concentration: 5000X in DMSO) in 20 mM Tris, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z

Nandy

Maranholkar

Crum

et al. 2023

IJMS

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Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of −33.0 kcal/mol and −32.7 kcal/mol, and −T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

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Section: Methodsmentioning

confidence: 99%