Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

The Importance of Location of Tryptophan Residues in Probing the Folding Pathway of Factor for Inversion Stimulation

Tryptophan residues in proteins can be used as an internal fluorophore to monitor protein conformational changes. Factor for inversion stimulation (FIS), a small homodimeric DNA binding protein, does not contain any tryptophan residues, but has four tyrosine residues that are located at positions 38, 51, 69, and 95. The folding of P61A FIS was followed by using its native intrinsic fluorophores. However, difficulties were encountered because tyrosine is not a strong fluorophore, and the tyrosine residues are located in different environments and involved in specific intra- or inter-molecular interactions. Due to the weak fluorescence signal it was first thought that the folding pathway of P61A FIS was two-state (N2⇔2U). The decrease in the slope of the denaturation curve (m-value) at increasing protein concentrations suggested that the equilibrium denaturation involved a three-state (N2 ⇔ I2 ⇔2U) mechanism, which was then confirmed by global fitting of the denaturation transitions. Therefore, a series of P61A/YxxW double mutants were made where each individual tyrosine residue in P61A FIS was mutated to a tryptophan residue. This enabled us to probe the conformational changes at one specific region of FIS. The urea and guanidine hydrochloride (GdnHCl) induced equilibrium denaturations of these mutants were monitored by circular dichroism (CD) and fluorescence. Global fitting was used to analyze the equilibrium denaturation data. Each mutant was able to detect the dimeric intermediate in the folding pathway differently. All the denaturation curves for the P61A/Y95W FIS mutant were concentration dependent and had a deceasing m-value which was evidence of an intermediate in the denaturation pathway. This mutant was able to homogenously destabilize the protein. In contrast, a clear intermediate was seen by the biphasic far UV CD denaturation curves of the P61A/Y69W FIS mutant. The first transition is due to the unfolding of the C-terminus and the second transition is caused by the denaturation of the dimeric intermediate. Unexpectedly, the dimeric intermediate is distinctly apparent in the P61A/Y38W FIS mutant, which happens to be the most stable mutant. The fluorescence denaturation curves were concentration independent, indicating that they are only detecting the first transition. The far UV CD denaturation curves were more decoupled and were able to probe the full denaturation process. Thus, these results emphasize the importance of the location of the fluorophore in determining what is seen in equilibrium denaturation experiments. Furthermore, the unpredictable data obtained with the different mutants demonstrate that it is difficult to anticipate the best location for a tryptophan residue to probe the folding and unfolding of proteins.

Virginia A. Muñiz*1
Sarah Boswell1
Derrick W. Meinhold1
Tawanna Childs1
Robert Osuna2
Wilfredo Colón1

1Dept of Chem. & Chemical Biology
Rensselaer Polytechnic Institute
110 8th Street
Troy, NY 12180

2Dept of Biological Sciences
SUNY at Albany
Albany, NY 12222

*Phone: (518) 276-4139
Fax: (518) 276-2851
Email: munizv@rpi.edu