GFP-linked zinc finger protein sp1: fluorescence study and implication for N-terminal zinc finger 1 as hinge finger

Abstract

The N-terminal zinc finger (zf) of Sp1 is referred to as the ‘hinge finger’, which connects the C-terminal DNA binding domain with the N-terminal activation domain. In this study, we investigated how a green fluorescent protein (GFP) linked to the N-terminal zinc finger is located spatially. The fluorescence resonance energy transfer technique and steady-state fluorescence anisotropy measurements indicate the results as follows: (1) In the binding to GC-box DNA, the geometry of the GFP domain of the GFP-linked Sp1 zinc finger is similar to that of the Ala-556→Arg mutant. (2) The GFP-linked Sp1 zinc finger is folded more compactly in the absence of DNA (hydrodynamic volume V=78.2 nm 3) and consequently alters the conformation at the GFP domain more extensively (Δ V=43.6 nm 3) upon DNA binding than the Ala-556→Arg mutant (99.5, 14.8 nm 3, respectively). These results implicate that the N-terminal ‘hinge finger’ moderates various interactions of the adjacent N-terminal regulation domain with other transcriptional factors as well as DNA binding and is essential for the function of Sp1 and/or Sp/XKLF family members.