Replacement of the active surface of a thermophile protein by that of a homologous mesophile protein through structure-guided 'protein surface grafting'.
Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.
Division of Protein Science and Engineering, Institute of Microbial Technology, Chandigarh 160 036, India.
Divya
D
Kapoor
Vijay
V
Kumar
Sanjeev K
SK
Chandrayan
Shubbir
S
Ahmed
Swati
S
Sharma
Manish
M
Datt
Balvinder
B
Singh
Subramanian
S
Karthikeyan
Purnananda
P
Guptasarma
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D045245:Rhodothermus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013499:Surface Properties; D013696:Temperature; D013816:Thermodynamics; D044366:Transition Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D044602:Cellulases