title: Detecting O2 binding sites in protein cavities.
abstract: Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in (1)H, (15)N, and (13)C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.
authors.affiliation: College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu 525-8577, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kitahara
authors.affiliation: Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Yoshimura
authors.affiliation: Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark.
authors.forename: Mengjun
authors.initials: M
authors.lastname: Xue
authors.affiliation: Biotechnology Research Institute for Drug Discovery, Advanced Industrial Science and Technology (AIST), 2-43 Aomi, Koto, Tokyo 135-0064, Japan.
authors.forename: Tomoshi
authors.initials: T
authors.lastname: Kameda
authors.affiliation: Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark.
authors.forename: Frans A A
authors.initials: FA
authors.lastname: Mulder
mesh_terms: D001665:Binding Sites; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011506:Proteins; D009113:Muramidase; D010100:Oxygen
references.citation: Nature. 1965 Jul 3;207(992):28-30
references.pmid: 5893727
references.citation: Biochemistry. 1995 Jul 11;34(27):8576-88
references.pmid: 7612599
references.citation: Proc Natl Acad Sci U S A. 2014 Sep 23;111(38):13846-51
references.pmid: 25201963
references.citation: J Magn Reson. 2002 Aug;157(2):181-9
references.pmid: 12323136
references.citation: Biophys J. 2007 Nov 15;93(10):3591-600
references.pmid: 17693478
references.citation: Langmuir. 2006 May 9;22(10):4620-8
references.pmid: 16649773
references.citation: J Mol Graph. 1990 Mar;8(1):52-6, 29
references.pmid: 2268628
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: J Mol Biol. 2000 Sep 29;302(4):979-89
references.pmid: 10993736
references.citation: J Am Chem Soc. 2002 Apr 24;124(16):4463-72
references.pmid: 11960476
references.citation: J Am Chem Soc. 2005 Apr 27;127(16):5826-32
references.pmid: 15839680
references.citation: Nature. 2011 Sep 1;477(7362):111-4
references.pmid: 21857680
references.citation: J Mol Graph. 1996 Feb;14(1):51-5, 29-32
references.pmid: 8744573
references.citation: Nat Struct Biol. 1996 Feb;3(2):178-87
references.pmid: 8564545
references.citation: J Am Chem Soc. 2002 Feb 20;124(7):1443-51
references.pmid: 11841314
references.citation: Nature. 1965 Nov 20;208(5012):760-2
references.pmid: 5868886
references.citation: Biophys J. 2004 Mar;86(3):1713-25
references.pmid: 14990499
references.citation: Biophys Chem. 2011 Jun;156(1):24-30
references.pmid: 21367514
references.citation: J Chem Phys. 2013 Mar 7;138(9):094503
references.pmid: 23485309
references.citation: J Am Chem Soc. 2003 Apr 2;125(13):3813-20
references.pmid: 12656614
references.citation: J Am Chem Soc. 2005 Aug 24;127(33):11676-83
references.pmid: 16104744
references.citation: J Phys Chem A. 2006 Jan 19;110(2):580-8
references.pmid: 16405330
references.citation: Biophys J. 2012 Feb 22;102(4):916-26
references.pmid: 22385863
references.citation: Biochemistry. 2012 Nov 27;51(47):9535-46
references.pmid: 23116341
references.citation: Biochemistry. 1973 Oct 9;12(21):4171-9
references.pmid: 4200894
references.citation: Biochemistry. 2000 Oct 17;39(41):12614-22
references.pmid: 11027141
references.citation: J Mol Biol. 1996 Aug 16;261(2):289-300
references.pmid: 8757295
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: J Mol Biol. 1993 Feb 5;229(3):747-69
references.pmid: 8433369
references.citation: J Mol Biol. 1980 Oct 5;142(4):531-54
references.pmid: 7463482
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14406-11
references.pmid: 18780783
references.citation: Biochemistry. 1983 Mar 29;22(7):1526-32
references.pmid: 6342662
references.citation: Nat Struct Biol. 2001 Nov;8(11):932-5
references.pmid: 11685237
references.citation: Nat Struct Biol. 1997 May;4(5):396-404
references.pmid: 9145111
references.citation: Biophys J. 2015 Jan 6;108(1):133-45
references.pmid: 25564860
references.citation: Biophys J. 2006 Sep 1;91(5):1844-57
references.pmid: 16751246
references.citation: J Phys Chem B. 2005 Sep 29;109(38):17795-8
references.pmid: 16853281
references.citation: Channels (Austin). 2013 May-Jun;7(3):182-93
references.pmid: 23545989
references.citation: Nature. 1992 Jan 23;355(6358):371-3
references.pmid: 1731252
references.citation: J Mol Biol. 2000 Sep 29;302(4):955-77
references.pmid: 10993735
references.citation: Nat Struct Biol. 1996 Jun;3(6):516-21
references.pmid: 8646537