Recognition and binding of a helix-loop-helix peptide to carbonic anhydrase occurs via partly folded intermediate structures.
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.
Department of Photochemistry and Molecular Sciences, Uppsala University, Uppsala, Sweden.
Martin
M
Lignell
Hans-Christian
HC
Becker
D000465:Algorithms; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D004735:Energy Transfer; D005453:Fluorescence; D005454:Fluorescence Polarization; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010713:Phosphatidylcholines; D011336:Probability; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012399:Rotation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013449:Sulfonamides / Q000737:chemistry; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014867:Water / Q000737:chemistry; D000096926:Benzenesulfonamides
D024402:Carbonic Anhydrase II; D010455:Peptides; D010713:Phosphatidylcholines; D013449:Sulfonamides; D014364:Tryptophan; D014867:Water; D000096926:Benzenesulfonamides; C496906:1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine