Recognition and binding of a helix-loop-helix peptide to carbonic anhydrase occurs via partly folded intermediate structures

Abstract

We have studied the association of a helix-loop-helix peptide scaffold carrying a benzenesulfonamide ligand to carbonic anhydrase using steady-state and time-resolved fluorescence spectroscopy. The helix-loop-helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, interpreted as differently interacting peptide/protein structures. We characterize these peptide/protein complexes as mostly bound but unfolded, bound and partly folded, and strongly bound and folded. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using the amplitudes of these times, we can correlate the lifetime groups with the corresponding fluorescence anisotropy component. The 23-ns rotational correlation time, which appears with the same amplitude as a 17-ns fluorescence lifetime, shows that the dansyl fluorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide/protein complex. A partly folded and partly hydrated interfacial structure is manifested in an 8-ns dansyl fluorescence lifetime and a 1.2-ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure, which allows segmental movement and has a higher degree of solvent exposure of dansyl. Indirect excitation of dansyl on the helix-loop-helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase shows that the helix-loop-helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that binding of the peptide to carbonic anhydrase involves a transition from a disordered to an ordered structure of the helix-loop-helix scaffold.

Figure 1

(a) Dansyl fluorescence spectra of the helix-loop-helix receptor (KE2D15-8). The pure KE2D15-8 spectrum (1 μM) shows the lowest fluorescence intensity and the most red-shifted emission maximum. Addition of carbonic anhydrase (0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.5, and 2.0 μM) increases the intensity and blue-shifts the emission maximum. Dansyl fluorescence intensity and changes in emission maximum level out after 1.0 μM. (b) Left axis: Concentration of the formed KE2D15-8/HCAII complex when 1 μM of KE2D15-8 is titrated with HCAII up to 2 μM. The concentration of the complex was estimated from the dansyl fluorescence intensity at 550 nm (solid squares). K_D was estimated at 1 nM by fitting a 1:1 binding model (Eq. 5) to the concentration of the complex (solid line). Right axis: Change of the dansyl emission maximum (open circles) as a function of concentration of carbonic anhydrase.

Figure 2

Fluorescence lifetime distributions of dansyl from KE2D15-8 complexed 1:1 with carbonic anhydrase (upper) and uncomplexed KE2D15-8 (lower).

Figure 3

(a) Emission spectrum of tryptophan (300–400 nm) and of dansyl (440–570 nm) upon excitation at 290 nm of 1 μM HCAII at increasing concentrations of KE2D15-8 (0, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 2.0 μM). (Inset) Tryptophan and dansyl emission intensities as a function of HCAII concentration. Note the much more rapid increase in dansyl emission during the binding phase (0–1 μM). (b) Overlapping absorption spectrum of 1μM KE2D15-8 (circles) and emission spectrum of 1μM HCAII (squares), mediating Förster energy transfer from tryptophan(s) in HCAII to dansyl on the helix-loop-helix.

Figure 4

(a) Dansyl fluorescence lifetime distributions of the KE2D15-8:HCAII complex when dansyl is excited through energy transfer from tryptophan(s) in the HCAII protein. The distribution lacks the 3.1-ns peak, found only when dansyl in the complex is excited directly at 335 nm (compare with Fig. 2). (b) Excitation of dansyl in the KE2D15-8/HCAII complex via energy transfer from tryptophans in HCAII results in a blue-shifted dansyl emission spectrum (solid line) relative to the directly excited dansyl spectrum at 335 nm (dashed line).