Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation.
The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA.
Sarah
S
Boswell
John
J
Mathew
Michael
M
Beach
Robert
R
Osuna
Wilfredo
W
Colón
D000595:Amino Acid Sequence; D002942:Circular Dichroism / Q000379:methods; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D035021:Factor For Inversion Stimulation Protein / Q000737:chemistry* / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D014508:Urea
D004268:DNA-Binding Proteins; D035021:Factor For Inversion Stimulation Protein; D014443:Tyrosine; D010649:Phenylalanine; D014508:Urea; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin