Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin.
Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu(9), Glu(194), and Glu(204), were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.
Sapra
K Tanuj
KT
Biotechnology Center, University of Technology, Dresden, Germany. sapra@biotec.tu-dresden.de
Doehner
Jana
J
Renugopalakrishnan
V
V
Padrós
Esteve
E
Muller
Daniel J
DJ
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001696:Biomechanical Phenomena; D005110:Extracellular Space / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D019617:Halobacterium salinarum; D007700:Kinetics; D009154:Mutation; D011489:Protein Denaturation; D013816:Thermodynamics
D018698:Glutamic Acid; D001436:Bacteriorhodopsins