Probing amino acid side chains of the integral membrane protein PagP by solution NMR: Side chain immobilization facilitates association of secondary structures
Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived 1H-13C magnetization in methyl groups and/or backbone amide 1H-15N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is poss...
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| Published in: | Biochimica et biophysica acta. Biomembranes Vol. 1866; no. 3; p. 184281 |
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| Main Authors: | , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Netherlands
Elsevier B.V
01-03-2024
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived 1H-13C magnetization in methyl groups and/or backbone amide 1H-15N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is possible to biosynthetically incorporate additional 1H-12C groups that possess long-lived magnetization using cost-effective partially deuterated or unlabeled amino acid precursors added to Escherichia coli growth media. This approach is applied to the outer membrane enzyme PagP in membrane-mimetic dodecylphosphocholine micelles.
We were able to obtain chemical shift assignments for a majority of side chain 1H positions in PagP using nuclear Overhauser enhancements (NOEs) to connect them to previously assigned backbone 1H-15N groups and newly assigned 1H-13C methyl groups. Side chain methyl-to-aromatic NOEs were particularly important for confirming that the amphipathic α-helix of PagP packs against its eight-stranded β-barrel, as indicated by previous X-ray crystal structures.
Interestingly, aromatic NOEs suggest that some aromatic residues in PagP that are buried in the membrane bilayer are highly mobile in the micellar environment, like Phe138 and Phe159. In contrast, Tyr87 in the middle of the bilayer is quite rigid, held in place by a hydrogen bonded network extending to the surface that resembles a classic catalytic triad: Tyr87-His67-Asp61. This hydrogen bonded arrangement of residues is not known to have any catalytic activity, but we postulate that its role is to immobilize Tyr87 to facilitate packing of the amphipathic α-helix against the β-barrel.
The solution NMR structure of bacterial outer membrane protein PagP (red) agrees with the X-ray crystal structure (blue) in the packing of the amphipathic helix against the β-barrel. An important structural feature that enables this packing is the immobilization of barrel residues via a network of hydrogen bonds comprised of D61, H67, and Y87 that superficially resembles a classic catalytic triad. [Display omitted]
•Selective deuteration of aromatic residues for solution 1H NMR of big proteins•Biosynthetic incorporation of deuterated aromatic precursors in E. coli•NOEs between methyl 1H-13C, backbone amide 1H-15N, and side chain 1H-12C•In the membrane, side chain immobilization facilitates protein packing. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 ObjectType-Review-3 content type line 23 |
| ISSN: | 0005-2736 1879-2642 1879-2642 |
| DOI: | 10.1016/j.bbamem.2024.184281 |