title: Quasithermodynamic contributions to the fluctuations of a protein nanopore.
abstract: Proteins undergo thermally activated conformational fluctuations among two or more substates, but a quantitative inquiry on their kinetics is persistently challenged by numerous factors, including the complexity and dynamics of various interactions, along with the inability to detect functional substates within a resolvable time scale. Here, we analyzed in detail the current fluctuations of a monomeric β-barrel protein nanopore of known high-resolution X-ray crystal structure. We demonstrated that targeted perturbations of the protein nanopore system, in the form of loop-deletion mutagenesis, accompanying alterations of electrostatic interactions between long extracellular loops, produced modest changes of the differential activation free energies calculated at 25 °C, ΔΔG(⧧), in the range near the thermal energy but substantial and correlated modifications of the differential activation enthalpies, ΔΔH(⧧), and entropies, ΔΔS(⧧). This finding indicates that the local conformational reorganizations of the packing and flexibility of the fluctuating loops lining the central constriction of this protein nanopore were supplemented by changes in the single-channel kinetics. These changes were reflected in the enthalpy-entropy reconversions of the interactions between the loop partners with a compensating temperature, TC, of ∼300 K, and an activation free energy constant of ∼41 kJ/mol. We also determined that temperature has a much greater effect on the energetics of the equilibrium gating fluctuations of a protein nanopore than other environmental parameters, such as the ionic strength of the aqueous phase as well as the applied transmembrane potential, likely due to ample changes in the solvation activation enthalpies. There is no fundamental limitation for applying this approach to other complex, multistate membrane protein systems. Therefore, this methodology has major implications in the area of membrane protein design and dynamics, primarily by revealing a better quantitative assessment on the equilibrium transitions among multiple well-defined and functionally distinct substates of protein channels and pores.
authors.affiliation: †Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States.
authors.forename: Belete R
authors.initials: BR
authors.lastname: Cheneke
authors.affiliation: ‡Institute for Cellular and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom.
authors.forename: Bert
authors.initials: B
authors.lastname: van den Berg
authors.affiliation: †Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States.|§Structural Biology, Biochemistry, and Biophysics Program, Syracuse University, 111 College Place, Syracuse, New York 13244-4100, United States.|∥Syracuse Biomaterials Institute, Syracuse University, 121 Link Hall, Syracuse, New York 13244, United States.
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008567:Membranes, Artificial; D008958:Models, Molecular; D058608:Nanopores / Q000648:ultrastructure*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002264:Carboxylic Acids; D026901:Membrane Transport Proteins; D008567:Membranes, Artificial; D011994:Recombinant Proteins