Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin.
A bioinformatics-based protein-engineering approach called consensus design led to the construction of a chimeric triosephosphate isomerase (TIM) protein called ccTIM (curated consensus TIM) which is as active as Saccharomyces cerevisiae TIM despite sharing only 58% sequence identity with it. The amino-acid sequence of this novel protein is as identical to native sequences from eukaryotes as to those from prokaryotes and shares some biophysical traits with a molten globular protein. Solving its crystal structure would help in understanding the physical implications of its bioinformatics-based sequence. In this report, the ccTIM protein was successfully crystallized using the microbatch-under-oil method and a full X-ray diffraction data set was collected to 2.2 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C2221, with unit-cell parameters a=107.97, b=187.21, c=288.22 Å. Matthews coefficient calculations indicated the presence of six dimers in the asymmetric unit, with an approximate solvent content of 46.2%.
Chemistry Department, IIT Bombay, Powai, Mumbai 400 076, India.
Venuka Durani
VD
Goyal
High Pressure and Synchrotron Radiation Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Pooja
P
Yadav
High Pressure and Synchrotron Radiation Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Ashwani
A
Kumar
High Pressure and Synchrotron Radiation Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Biplab
B
Ghosh
High Pressure and Synchrotron Radiation Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Ravindra D
RD
Makde
D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D010802:Phylogeny; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics*
D014305:Triose-Phosphate Isomerase
consensus design; molten globule; protein engineering; triosephosphate isomerase