Here we report on the structural features of the triple mutant E9Q/E194Q/E204Q (3Glu) of bacteriorhodopsin (bR) by combining experimental and molecular dynamics (MD) approaches. In 3Glu mutant, Glu9, Glu194 and Glu204 residues located at the extracellular side of the protein were mutated all together to glutamines. UV-Vis and DSC experiments served as diagnostic tools for monitoring the resistance against thermal stress of the active site and the tertiary structures of the 3Glu. The analyses of the UV-Vis thermal difference spectra demonstrate that the spectral forms at room temperature and the thermal unfolding path differ in the WT and the 3Glu. Even these spectral differences, the thermal unfolding of the active site occurs at rather similar melting temperatures in both proteins. A noteworthy consequence of the mutations is the altered 2D packing revealed by the lack of the pre-transition peak in DSC traces of 3Glu mutant, previously detected in WT and the corresponding single mutants. The infrared spectroscopy data agree with the loss of paracrystalinity illustrating a substantial conversion of αII to αI helical conformation in the 3Glu mutant. MD simulations show higher dynamics flexibility of most of EC regions of 3Glu, which may account for somewhat lower tertiary structural stability of the mutated protein. Finally, the hydrogen bond analysis reveals that the mutated Glu194 and Glu204 residues create about 50% less hydrogen bonds with water molecules compared to WT bR. These results exemplify the role of the water hydrogen-bonding network for structural integrity and conformational flexibility of bR. This article is protected by copyright. All rights reserved.