Conformational changes of maize and wheat NADP-malic enzyme studied by quenching of protein native fluorescence.
Quenching of tryptophan fluorescence of maize and wheat NADP-malic enzyme by KI and acrylamide was studied after denaturating proteins with guanidine hydrochloride, and subjecting them to different pH values or temperatures. Protein unfolding by guanidine hydrochloride resulted in a red shift of the fluorescence spectrum, providing further support for the motion that several of the tryptophan residues evolved from an apolar to a polar environment. Protein denaturation was accompanied by an increase in the effective dynamic quenching constant values and by loss of the enzyme's activities. Thermal denaturation gave results consistent with the ones observed for chemical denaturation suggesting that a putative intermediate is involved in the denaturation process. Finally, exposure of both enzymes at various pH values allowed us to infer the number of accessible tryptophan residues in the different oligomeric conformations. The results suggest that the aggregation process seems to be different for each enzyme. Thus, as the maize enzyme associated from monomer to tetramer, one tryptophan residue would change from a polar to an apolar environment, while the association of the wheat enzyme would cause that two tryptophan residues to be excluded from quenching. Hitherto, quenching of the tryptophan fluorescence provides a good tool for studying conformational changes of proteins. The future availability of the crystal structures of plant NADP-malic enzymes will offer a good validation point for our model and the technology used.
Spampinato
Claudia P
CP
Centro de Estudios Fotosintéticos y Bioquímicos (CONICET-Fund. M. Lillo), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina. spampinato@cefobi.gov.ar
Ferreyra
María Lorena Falcone
ML
Andreo
Carlos S
CS
D000595:Amino Acid Sequence; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D014364:Tryptophan; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D019791:Guanidine