Potent efficacy of an IgG-specific endoglycosidase against IgG-mediated pathologies.

Endo-β-N-acetylglucosaminidases (ENGases) that specifically hydrolyze the Asn297-linked glycan on immunoglobulin G (IgG) antibodies, the major molecular determinant of fragment crystallizable (Fc) γ receptor (FcγR) binding, are exceedingly rare. All previously characterized IgG-specific ENGases are multi-domain proteins secreted as an immune evasion strategy by Streptococcus pyogenes strains. Here, using in silico analysis and mass spectrometry techniques, we identified a family of single-domain ENGases secreted by pathogenic corynebacterial species that exhibit strict specificity for IgG antibodies.

Disordered Domain Shifts the Conformational Ensemble of the Folded Regulatory Domain of the Multidomain Oncoprotein c-Src.

c-Src kinase is a multidomain non-receptor tyrosine kinase that aberrantly phosphorylates several signaling proteins in cancers. Although the structural properties of the regulatory domains (SH3-SH2) and the catalytic kinase domain have been extensively characterized, there is less knowledge about the N-terminal disordered region (SH4UD) and its interactions with the other c-Src domains. Here, we used domain-selective isotopic labeling combined with the small-angle neutron scattering contrast matching technique to study SH4UD interactions with SH3-SH2.

Yeast Surface Display: New Opportunities for a Time-Tested Protein Engineering System.

Yeast surface display has proven to be a powerful tool for the discovery of antibodies and other novel binding proteins and for engineering the affinity and selectivity of existing proteins for their targets. In the decades since the first demonstrations of the approach, the range of yeast display applications has greatly expanded to include many different protein targets and has grown to encompass methods for rapid protein characterization. Here, we briefly summarize the development of yeast display methodologies and highlight several selected examples of recent applications to timely and challenging protein engineering and characterization problems.

Identifying Stable Fragments of Arabidopsis thaliana Cellulose Synthase Subunit 3 by Yeast Display.

Determining structures of large, complex proteins remains challenging, especially for transmembrane proteins, as the protein size increases. Arabidopsis thaliana cellulose synthesis complex is a large, multimeric complex located in the plant cell membrane that synthesizes cellulose microfibrils in the plant cell wall. Despite the biological and economic importance of cellulose and therefore cellulose synthesis, many aspects of the cellulase synthase complex (CSC) structure and function are still unknown.

Fine-tuning sortase-mediated immobilization of protein layers on surfaces using sequential deprotection and coupling.

Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer-by-layer protein attachment. Here, we report a technique for controlling enzyme-mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase-cleavable peptide sequence was inserted at the N-terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A-mediated protein immobilization on the surface layer-by-layer.