Traditional protein site-directed mutagenesis has served biochemists for many years but is lacking when one seeks to construct libraries of mutants such as in alanine scanning. In protein engineering experiments, protein libraries are redundant mixtures requiring more screening or selection than would be necessary from a """"""""perfect"""""""" mixture. This proposal seeks to create a simple, fast, and effective way to perform scanning codon mutagenesis throughout any protein coding sequence. Coupled with a reading frame selection, this will allow the rapid production of collections of proteins that contain substitutions of a defined set of amino acids, including those that contain unnatural functional groups. The proposed studies will i)develop a Mu transposon-based method for the random insertion and deletion of in-frame codon mutations from an open reading frame, ii) verify this methodology by performing whole-gene alanine scanning on an a counterselectable enzyme expressed in E. coli iii) generate a series of """"""""smart"""""""" 2-lactamase enzyme libraries which are rich in disulfide bonds or charged amino acids and screen those libraries for improved thermostablity, and iv)use this technology to singly incorporate photo-crosslinking amino acids at every possible position throughout a multi-protein complex allowing subsequent analysis by mass-spectroscopy. PUBLIC HEALTH RELEVENCE: This proposal will develop new protein engineering technology that provides access to improved biocatalysts and therapeutic proteins. Furthermore it will provide a tool for structure-function studies on large protein complexes and membrane proteins, both of which are difficult to study using currently available methods.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Virginia Commonwealth University
Schools of Arts and Sciences
United States
Zip Code
Castañeda, Carlos A; Dixon, Emma K; Walker, Olivier et al. (2016) Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins. Structure 24:423-36
Liu, Shu-su; Wei, Xuan; Dong, Xue et al. (2015) Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations. BMC Biochem 16:17
Knight, William A; Cropp, T Ashton (2015) Genetic encoding of the post-translational modification 2-hydroxyisobutyryl-lysine. Org Biomol Chem 13:6479-81
Liu, Jia; Cropp, T Ashton (2013) Rational protein sequence diversification by multi-codon scanning mutagenesis. Methods Mol Biol 978:217-28
Liu, Jia; Cropp, T Ashton (2012) A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons. Protein Eng Des Sel 25:67-72
Liu, Jia; Cropp, T Ashton (2012) Experimental methods for scanning unnatural amino acid mutagenesis. Methods Mol Biol 794:187-97
Castaneda, Carlos A; Liu, Jia; Kashyap, Tanuja R et al. (2011) Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups. Chem Commun (Camb) 47:2026-8
Castaneda, Carlos; Liu, Jia; Chaturvedi, Apurva et al. (2011) Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme. J Am Chem Soc 133:17855-68
Liu, Jia; Castaneda, Carlos A; Wilkins, Bryan J et al. (2010) Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids. Bioorg Med Chem Lett 20:5613-6
Daggett, Kelly A; Layer, Mark; Cropp, T Ashton (2009) A general method for scanning unnatural amino acid mutagenesis. ACS Chem Biol 4:109-13