title: Infrared and fluorescence assessment of the hydration status of the tryptophan gate in the influenza A M2 proton channel.
abstract: The M2 proton channel of the influenza A virus has been the subject of extensive studies because of its critical role in viral replication. As such, we now know a great deal about its mechanism of action, especially how it selects and conducts protons in an asymmetric fashion. The conductance of this channel is tuned to conduct protons at a relatively low biologically useful rate, which allows acidification of the viral interior of a virus entrapped within an endosome, but not so great as to cause toxicity to the infected host cell prior to packaging of the virus. The dynamic, structural and chemical features that give rise to this tuning are not fully understood. Herein, we use a tryptophan (Trp) analog, 5-cyanotryptophan, and various methods, including linear and nonlinear infrared spectroscopies, static and time-resolved fluorescence techniques, and molecular dynamics simulations, to site-specifically interrogate the structure and hydration dynamics of the Trp41 gate in the transmembrane domain of the M2 proton channel. Our results suggest that the Trp41 sidechain adopts the t90 rotamer, the χ2dihedral angle of which undergoes an increase of approximately 35° upon changing the pH from 7.4 to 5.0. Furthermore, we find that Trp41 is situated in an environment lacking bulk-like water, and somewhat surprisingly, the water density and dynamics do not show a measurable difference between the high (7.4) and low (5.0) pH states. Since previous studies have shown that upon channel opening water flows into the cavity above the histidine tetrad (His37), the present finding thus provides evidence indicating that the lack of sufficient water molecules near Trp41 needed to establish a continuous hydrogen bonding network poses an additional energetic bottleneck for proton conduction.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. gai@sas.upenn.edu.
authors.forename: Beatrice N
authors.initials: BN
authors.lastname: Markiewicz
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143, USA. william.degrado@ucsf.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lemmin
authors.affiliation: Ultrafast Optical Processes Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Wenkai
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Ismail A
authors.initials: IA
authors.lastname: Ahmed
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143, USA. william.degrado@ucsf.edu.
authors.forename: Hyunil
authors.initials: H
authors.lastname: Jo
authors.affiliation: Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania 19122, USA.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Fiorin
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. gai@sas.upenn.edu and Ultrafast Optical Processes Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Troxler
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143, USA. william.degrado@ucsf.edu.
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. gai@sas.upenn.edu and Ultrafast Optical Processes Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Gai