title: Membrane-Associated Conformation of HIV-1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer.
abstract: In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane.
authors.affiliation: †Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115-5000, United States.
authors.forename: Gregory F
authors.initials: GF
authors.lastname: Pirrone
authors.affiliation: ‡Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Emert-Sedlak
authors.affiliation: †Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115-5000, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wales
authors.affiliation: ‡Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
authors.affiliation: §Bioenergy and Defense Technologies, Sandia National Laboratories, Albuquerque, New Mexico 87185, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kent
authors.affiliation: †Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115-5000, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
mesh_terms: D041961:Deuterium Exchange Measurement; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D013058:Mass Spectrometry; D008566:Membranes / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005229:Fatty Acids, Monounsaturated; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D006859:Hydrogen; C054211:9-tetradecenoic acid