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Abstract
Site-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.
Highlights
Modification of a unique cysteine residue with a maleimide or iodoacetamide conjugated probe is a widely used method of producing site- labeled proteins
We reasoned that amino acid substitutions derived from close MmCpn homologs would be well tolerated by this chaperonin
To this end we amassed 2981 group II chaperonin sequences from the NCBI non-redundant protein sequence database using BLAST17. Each of these extant chaperonin sequences was aligned to the wild type (WT) MmCpn sequence using the program Water from the EMBOSS Suite[18]
Summary
Modification of a unique cysteine residue with a maleimide or iodoacetamide conjugated probe is a widely used method of producing site- labeled proteins These two chemistries have a long history in the literature starting from the first description of the reaction between iodoacetamide and thiols in 19331,2. A rational method to produce active cysteine-less protein variants would be quite desirable to contemporary protein science Along these lines, we sought to generate a cysteine-less variant of a well studied group II chaperonin, MmCpn. Chaperonins are ATP-driven components of the cellular protein folding machinery and are essential in all free-living organisms. The group II chaperonins are found exclusively in the archaeal and eukaryotic cytosol[12] They are a family of ATP-driven hexadecameric chaperones with two stacked, eight membered rings per complex. We will discuss the homohexadecameric group II chaperonin from Methanococcus maripaludis, MmCpn, which has proven itself to be a very useful model group II chaperonin
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