Tolbert Lab: Protein Chemistry

Protein Chemistry

A major challenge of protein chemistry is the specific chemical modification of proteins. Proteins are linear chains of amino acids which can have hundreds of similar chemical moieties strung out along their polypeptide backbones. Distinguishing a single chemical site on a protein can be a very difficult task, but has certain advantages over non-specific chemical modification. Random protein modification produces mixtures of modified proteins that have different degrees of modification and physical behaviors, and are not suited for precise biochemical and biophysical studies or medical applications. Specific protein modification, on the other hand, produces a homogeneously modified protein product which is well suited for biochemical studies and development of protein therapeutics.

Site specific protein modification can be accomplished through chemoselective reactions which are selective for specific chemical moieties on proteins. Chemoselective ligations such as those reactions shown below (triazole, oxime, hydrazone, and thiazolidine formation, and also native chemical ligation) take advantage of chemical moieties which occur rarely in proteins, or which can be selectively generated at specific sites on proteins. For example, oxime, hydrazone, and thiazolidine formation all take advantage of the chemical reactivity of aldehydes, which rarely occur in proteins, but can be generated on proteins which contain N-terminal serine or threonine residues through periodate oxidation. Much of our previous work in protein chemistry has utilized native chemical ligation/expressed protein ligation techniques.

Native chemical ligation/expressed protein ligation techniques utilize the unique chemical reactivity of N-terminal cysteines and thioesters. Both thioesters and N-terminal cysteines can be generated on expressed proteins to allow N- or C-terminal protein modification.

Generation of C-terminal thioesters. C-terminal thioesters can be generated on proteins by utilizing protein splicing elements called inteins. Inteins excise themselves from between two exterior sequences (exteins) breaking the polypeptide backbone in two places and then joining the two exteins together. Mutated inteins which undergo only the first step of protein splicing, breaking of the polypeptide backbone and formation of a thioester, can be used to form thioesters in expressed proteins. Modification agents containing N-terminal cysteines can then be used to modify the C-terminus of the expressed protein.

A wide variety of chemical labels and modification agents can be incorporated onto the C-terminus of proteins expressed as intein fusion proteins. Examples of thioester reagents used for the modification of maltose binding protein (MBP) with fluorescent labels, metal chelators, biotin, nucleotides, and glycosylation are shown below.

Generation of N-terminal cysteines. In order to generate an N-terminal cysteine from expressed proteins, the N-terminal methionine required for initiation of protein translation must be removed. We utilize a TEV protease cleavable linker strategy to generate N-terminal cysteines. This has the advantage of allowing an affinity tag to be placed N-terminal to the cleavage site which can be utilized for affinity purification and then removed during TEV protease cleavage.

The tobacco etch virus (TEV) protease is a highly selective cysteine protease. It has a 7 amino acid recognition site and usually cleaves between a glutamine and a serine or glycine residue. Replacement of the serine or glycine residue with a cysteine residue results in a mutated cleavage site which is still cleaved by TEV protease, but which generates an N-terminal cysteine upon cleavage. One major advantage of TEV protease is that it seldom cleaves proteins non-specifically, as demonstrated by the GPRT cleavage to the right.

TEV protease cleavable affinity-tagged proteins can be utilized for protein labeling and modification. Below is an example of glycosylation of human interleukin-2 (IL2) with a glycopeptide thioester.

We are currently pursuing a variety of chemoselective approaches to labeling and modification of expressed peptides and proteins, with the goal of applying these protein chemistry techniques to the modification of bioactive peptides and proteins.