title: The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS.
abstract: Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N(2) ⇆ I(2) ⇆ 2U) process involving a dimeric intermediate (I(2)). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N(2) ⇆ I(2) transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.
authors.affiliation: Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
authors.forename: Virginia A
authors.initials: VA
authors.lastname: Muñiz
authors.forename: Saipraveen
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Boswell
authors.forename: Derrick W
authors.initials: DW
authors.lastname: Meinhold
authors.forename: Tawanna
authors.initials: T
authors.lastname: Childs
authors.forename: Robert
authors.initials: R
authors.lastname: Osuna
authors.forename: Wilfredo
authors.initials: W
authors.lastname: Colón
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035021:Factor For Inversion Stimulation Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D014443:Tyrosine; D014364:Tryptophan