Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single-tryptophan mutants.
Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-A crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid.
Pattanaik
Priyaranjan
P
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Ravindra
Gudihal
G
Sengupta
Chandana
C
Maithal
Kapil
K
Balaram
Padmanabhan
P
Balaram
Hemalatha
H
D000818:Animals; D002942:Circular Dichroism; D005453:Fluorescence; D006016:Glycolates / Q000378:metabolism / Q000494:pharmacology; D019791:Guanidine / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D006016:Glycolates; D014364:Tryptophan; D014508:Urea; D014305:Triose-Phosphate Isomerase; C013193:phosphoglycolate; D019791:Guanidine