Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Giulia Di Rocco,Monica Caselli,Marco Sola,Antonio Ranieri,Fabrizio Bernini,Glauco Ponterini,Carlo Augusto Bortolotti,Marco Borsari,Gianantonio Battistuzzi,Ilaria Martinelli

doi:10.1515/zpch-2015-0749

Abstract

Abstract The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes.

Highlights

Intrinsic protein fluorescence represents a rich source of structural information [1,2,3]
We have studied the influence that binding of Cu2+, Ni2+, Zn2+ and Cr3+ ions to a C-terminal 6xHis-tag exerts on steady-state and time-resolved intrinsic fluorescence of wild type Pseudomonas aeruginosa azurin, as well as that of its R129W and W48A/R129W variants
Trp48 and Trp129 emissions of WT, R129W and W48AR129W azurins are quenched as a consequence of metal-ion binding to C-terminal fused 6xHis-tags

Summary

Introduction

Intrinsic protein fluorescence represents a rich source of structural information [1,2,3]. G., drug/target interaction studies or model experiments aimed at unraveling the biological interactors of a key protein Such structural changes can be monitored in in-vitro experiments and can be identified if the observed changes in the overall fluorescence response of the recombinant protein can be assigned to specific tryptophan residues [2, 3]. Because tryptophan is the lowest excitationenergy chromophore in proteins, energy transfer from tryptophan residues requires introduction of external quenchers In this contribution, we propose to take advantage of the widespread attachment of polyhistidine tags to recombinant proteins for purification purposes to increase the number of tryptophan quenching paths in a protein of interest. We show that this is readily achieved in three engineered his-tagged azurins by complexation of several bipositive metal ions at a 6xHis-tag

Objectives

Results

Conclusion