Unraveling multistate unfolding of pig kidney fructose‐1,6‐bisphosphatase using single tryptophan mutants

Abstract

Pig kidney fructose‐1,6‐bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride‐induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yañez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956–6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1–C2 or the C1–C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size‐exclusion chromatographic profiles and 1‐anilinonaphthalene‐8‐sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild‐type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum ∼ 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose‐1,6‐bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a ‘phase diagram’ reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1–C2 interface.

The Helix Located between the Two Domains of a Mip-like Peptidyl-Prolyl cis–trans Isomerase Is Crucial for Its Structure, Stability, and Protein Folding Ability

“…The phase diagram, a qualitative tool, has long been employed to identify the hidden unfolding–refolding intermediates of proteins. − Such a tool appeared to be very useful particularly when there is the lack of coincidence of the unfolding curves of proteins. To verify whether rFKBP22I3, rFKBP22I6, and rFKBP22D30 unfold with the formation of one or more intermediates, we constructed phase diagrams for all these proteins by plotting their Trp fluorescence intensity values at 320 nm versus the respective Trp fluorescence intensity values at 365 nm.…”

Effect of polyethylene glycols on the alkaline-induced molten globule intermediate of bovine serum albumin

“…The 'phase diagram' method, initially described by Burstein for the analysis of fluorescence data, is extremely sensitive for the detection of any intermediate states [17][18][19]. The essence of this method is to build up the diagram of I ( 1 ) versus I ( 2 ), where I ( 1 ) and I ( 2 ) are the fluorescence intensity values measured at wavelengths 1 and 2 under different experimental conditions when a protein is undergoing structural transformations.…”

Proline substitutions in a Mip-like peptidyl-prolyl cis-trans isomerase severely affect its structure, stability, shape and activity

“…To validate the above proposition, I 320 (the Trp fluorescence intensity at 320 nm) values of these proteins were plotted against their I 365 (Trp fluorescence intensity at 365 nm) values. Such plots are usually generated to recognize the hidden unfolding/refolding intermediates of proteins [54] , [55] , [56] . The plots yielded by V72P and A82P diverge from the linearity at urea concentrations of above 3 M ( Fig.…”