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.
Kapoor
Divya
D
Division of Protein Science and Engineering, Institute of Microbial Technology, Chandigarh 160 036, India.
Kumar
Vijay
V
Chandrayan
Sanjeev K
SK
Ahmed
Shubbir
S
Sharma
Swati
S
Datt
Manish
M
Singh
Balvinder
B
Karthikeyan
Subramanian
S
Guptasarma
Purnananda
P
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