In Situ Cyclization of Proteins (INCYPRO): Cross-Link Derivatization Modulates Protein Stability

Abstract

Protein macrocyclization represents a very efficient strategy to increase the stability of protein tertiary structures. Here, we describe a panel of novel C3-symmetric tris-electrophilic agents and their use for the cyclization of proteins. These electrophiles are reacted with a protein domain harboring three solvent-exposed cysteine residues, resulting in the in situ cyclization of the protein (INCYPRO). We observe a clear dependency of cross-linking rates on the electrophilicity. All nine obtained cross-linked protein versions show considerably increased thermal stability (up to 29°C increased melting temperature) when compared to that of the linear precursor. Most interestingly, the degree of stabilization correlates with the hydrophilicity of the cross-link. These results will support the development of novel cross-linked proteins and enable a more rational design process.

Protein Macrocyclization for Tertiary Structure Stabilization

“…In particular, INCYPRO provides straight‐forward access to diverse linker structures. [81] In general, it can be expected that more cyclization strategies will be developed which allow the simultaneous introduction of an additional functionality (e. g. PEG chains [84] or photo‐switchable moieties[ 85 , 86 ]).…”

“… [80] To facilitate the stabilization of natural tertiary structures, larger and water‐soluble tris‐electrophilic agents have been developed and used for the in situ cyclization of proteins (INCYPRO). [ 50 , 81 ] Here, three surface‐exposed cysteines are introduced ( 23 , Figure 6 a), preferably within different secondary structure elements. [ 50 , 81 ] Initially, a hydrophilic tris‐amine core ( Z1 ) was decorated with chloroacetamide ( 24 a )[ 82 , 83 ] and used to generate a bicyclic version of Sortase A.…”

“…[ 50 , 81 ] Here, three surface‐exposed cysteines are introduced ( 23 , Figure 6 a), preferably within different secondary structure elements. [ 50 , 81 ] Initially, a hydrophilic tris‐amine core ( Z1 ) was decorated with chloroacetamide ( 24 a )[ 82 , 83 ] and used to generate a bicyclic version of Sortase A. Notably, the INCYPRO reaction was selective for the three introduced cysteines tolerating the presence of an active‐site cysteine.…”

“…Later, a library of crosslinkers was composed using three different electrophiles ( 24 a ‐ 24 c , Figure 6 b) which were combined with three different C 3 ‐symmetric core structures ( Z1 ‐ Z3 , Figure 6 c). [81] The resulting nine tris‐electrophilic crosslinkers have then been used to cyclize the human KIX domain. All crosslinkers furnished bicyclic KIX domains with increased stability ( T m increase of 19–29 °C), with hydrophilic crosslink Z1 / 25 a providing the most pronounced stabilizing effect.…”

Automated Design of Macrocycles for Therapeutic Applications: From Small Molecules to Peptides and Proteins

“…[79][80][81] Recently, it was demonstrated that this chemistry can also be applied to the in situ cyclization of proteins. [82][83][84] For two proteins, Staphylococcus aureus sortase A (SrtA) and the KIX domain from the human CREB binding protein, cysteine residues were introduced in three surface-exposed positions and incubated with a triselectrophilic crosslinker to generate bicyclic enzymes with high tolerance towards thermal and chemical stress. 84 Given the three-dimensional (3D) structure of a linear peptidic or non-peptidic molecule as starting point, the first goal of cyclization is to maintain the conformation of the bioactive region that is relevant for biological recognition and affinity, and introduce linkers where interactions are either not affected or even further improved.…”