Cavity-creating mutation at the dimer interface of Plasmodium falciparum triosephosphate isomerase: restoration of stability by disulfide cross-linking of subunits.
Disulfide engineering across subunit interfaces provides a means of inhibiting dissociation during unfolding of multimeric enzymes. Two symmetry-related intersubunit disulfide bridges were introduced across the interface of the dimeric enzyme triosephosphate isomerase from Plasmodium falciparum. This was achieved by mutating a tyrosine residue at position 74 at the subunit interface to a cysteine, thereby enabling it to form a covalent cross-link with a pre-existing cysteine at position 13 of the other subunit. The wild-type enzyme (TIMWT) and the oxidized (Y74Cox) and reduced (Y74Cred) forms of the mutant have similar enzymatic activity, absorption, and fluorescence spectra. All three proteins have similar far-UV CD spectra. The Y74Cred shows a distinct loss of near-UV CD. Thermal precipitation studies demonstrate that TIMWT and Y74Cox have very similar Tm values (Tm approximately 60 degreesC) whereas Y74Cred is surprisingly labile (Tm approximately 38 degreesC). The Y74C mutant results in the creation of a large cavity (approximately 100 A3) at the dimer interface. The crystal structure for the oxidized form of Y74C mutant, crystallized in the presence of low concentrations of dithiothreitol, reveals an asymmetric dimer containing a disulfide bridge at one site and a reduced dithiol cysteine at the other. The crystal structure of the mutant offers insights into the destabilization effects of the interfacial cavities and the role of disulfide tethering in restoring protein stability.
Gopal
B
B
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Ray
S S
SS
Gokhale
R S
RS
Balaram
H
H
Murthy
M R
MR
Balaram
P
P
D000818:Animals; D011232:Chemical Precipitation; D002942:Circular Dichroism; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics*; D014443:Tyrosine / Q000235:genetics
D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; D014443:Tyrosine; D014305:Triose-Phosphate Isomerase; D003545:Cysteine