Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.

Background:The mechanism by which antibody constant region alters fine specificity is unknown. Results: Different constant regions were found to change electronic and chemical properties of the antigen-binding site. Conclusion: Constant regions can affect the energy landscape of the variable region. Significance: These results are potentially critical for understanding fast, correct immune responses at the systems level and for future immunotherapy development.

Split inteins are a class of naturally occurring proteins that carry out protein splicing in trans. The chemical mechanism of protein trans-splicing is well-understood and has been exploited to develop several powerful protein engineering technologies. Split intein chemistry is preceded by efficient molecular recognition between two protomers that become intertwined in their bound state. It is currently unclear how this unique topology is achieved upon fragment association. Using biophysical techniques in conjunction with protein engineering methods, including segmental isotopic labeling, we show that one split intein fragment is partly folded, while the other is completely disordered. These polypeptides capture each other through their disordered regions and form an ordered intermediate with native-like structure at their interface. This intermediate then collapses into the canonical intein fold. This mechanism provides insight into the evolutionary constraints on split intein assembly and should enhance the development of split intein-based technologies.

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Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoelike pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of inteinbased technologies.Protein splicing is a posttranslational modification, a multistep autoprocessing event, where intervening proteins (inteins) self-excise themselves from the precursors followed by the ligation of external proteins (exteins) (1). Once thought anomalies, inteins have since been found in all domains of life. Beyond their biological context, inteins are particularly intriguing as their capacity to process the polypeptide backbone makes them useful tools for protein engineering. Despite the fact that all inteins share the same fold and have highly conserved sequence motifs in their active sites, inteins have surprisingly different splicing efficiencies, and their extein sequence preferences differ dramatically. Here, we review studies on intein structure and dynamics that shed light on their complex conserved fold and their divergent efficiency and substrate specificity. The Intein FoldThe conserved horseshoe-like fold of inteins (Fig. 1A) has been seen in all intein structures solved by NMR and x-ray crystallography (2-11). The fold comprises primarily ␤-sheets, loops, and two short helices and has pseudo two-fold symmetry (Fig. 1B). Given this symmetry, it has been proposed that this fold arose due to a gene duplication event of some parent protein (6). The intein fold has three remarkable features: 1) the topology is complex, involving multiple passes of the polypeptide chain back and forth between the symmetry-related halves (Fig. 1B); 2) the extein-bearing termini of the intein are brought in close proximity (Ͻ10 Å) for splicing; and 3) multiple protease-like active sites composed of conserved sequence motifs are built around these termini to carry out each of the chemical steps involved in protein splicing. Folding of an intein is coupled to the reaction; the initial structure facilitates the first step of splicing, and thereafter each splicing step causes local conformational changes, affecting the fold of the catalytic apparatus and hence affecting the reaction coordinate and shifting the equilibrium positi...