NMR Structure of Mistic, a Membrane-Integrating Protein for Membrane Protein Expression

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NMR Structure of Mistic, a Membrane-Integrating
Protein for Membrane Protein Expression

• By Niloufar Safvati

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Structure Determination of Membrane Proteins

• Structural determination of soluble proteins has
  minimal restraints
• Structural determination of Membrane Proteins,
  however, has a couple of restraints
• 1. Production of high enough yield of protein
• 2. Crystallization

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Characteristics of an ideal fusion partner that
is specialized in producing recombinant IM
proteins

• An ideal fusion partner should
• autonomously traffic its cargo to the membrane,
  bypassing the translocon and associated toxicity
  issues
• retain the characteristics of other successful
  fusion partner proteins, including relatively
  small size, in vivo folding, and high stability.

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NMR Spectroscopy

• Can be used as an alternative method to
  crystallization
• NMR structure determination of IM proteins has
  been established only for very small,
  structurally simplistic IM proteins and for outer
  membrane bacterial porins
• New techniques for determining the
  characteristics of alpha helical IM proteins are
  therefore necessary

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What is Mistic?

• Mistic is a Bacillus subtilis integral membrane
  protein that folds into the membrane without the
  help of a translocon
• Mistic stands for Membrane-Integrating Sequence
  for Translation of Integral Membrane protein
  Constructs
• It consists of 110-amino acids (13kD)

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Why study Mistic?

• When recombinantly expressed in E. coli, Mistic
  associates tightly with the bacterial membrane.
• Surprisingly, Mistic is highly hydrophilic
• Mistic has most of the characterizations for
  being an ideal partner in the production of
  high-yields of integral membrane proteins

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Mistic Characterizations

• The in vivo topology of Mistic in E. coli was
  analyzed by evaluating the accessibility of an
  array of monocysteine mutants to the
  membrane-impermeable thiol biotinylating reagent
  3-(N-maleimidopropinyl) biocytin (MPB).
• In addition to the single naturally occurring
  cysteine (residue 3), cysteine mutations were
  introduced individually at the C terminus
  (residue 110) and in predicted loop regions at
  positions 30, 58, and 88, with the naturally
  occurring cysteine mutated to valine.
• Result
• This experiment revealed a well- exposed
  periplasmic C terminus. The lack of reactivity of
  the other locations indicates that they are
  either intracellular or membrane-embedded in
  Mistics native conformation.

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Only Glu110 at the C terminus is well exposed
periplasmically
Primary sequence of Mistic
Orange monocysteine probing residues
Green structural disruption mutants
Gray cloning artifact residues
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Secondary Structure of Mistic

• The secondary structure of Mistic was analyzed
  through NMR spectroscopy.
• The primary sequence was given backbone
  assignments which includes
• 1. The use of Transverse Relaxation
  Optimized Spectroscopy (TROSY)
• 2. The use of Nuclear Overhauser Effect
  Spectroscopy (NOESY)
• Result
• The 13Calpha chemical shift deviation from
  random coil values, the observed NOE pattern, and
  slow 1HN exchange with solvent strongly indicate
  the presence of four helices comprising residues
  8 to 22, 32 to 55, 67 to 81, and 89 to 102.

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Alpha Helices and Beta-sheets
Blue-chemical shifts in 0 mM K Green-chemical
shifts in 100 mM K

• Values larger than 1.5 ppm are indicative of an
  a-helical secondary structure
• Values smaller than -1.5 ppm are indicative of
  ß-sheet secondary structure.

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Transverse relaxation optimized spectroscopy
(TROSY)

• The NMR signal of large molecules has shorter
  transverse relaxation times compared to smaller
  molecules and therefore decays faster, leading to
  line broadening in the NMR spectrum which gives
  poor resolution and makes it difficult to analyze
  the molecule.
• The TROSY experiment is designed to choose the
  component for which the different relaxation
  mechanisms have almost cancelled, leading to a
  single, sharp peak in the spectrum. This
  significantly increases both spectral resolution
  and sensitivity leading to better results.

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Transverse Relaxation Optimized Spectroscopy
(TROSY)
Fernandex and Wider, Current Opinion in
Structural Biology 2003, 13570-580
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Nuclear Overhauser Effect Spectroscopy (NOESY)

• The Nuclear Overhauser Effect (NOE) is the
  transfer of nuclear spin polarization from one
  spin to another and is shown through NMR
  spectroscopy.
• All atoms that are in proximity to each other
  give a NOE.
• The distance can be derived from the observed
  NOEs, so that the precise, three-dimensional
  structure of the molecule can be reconstructed.

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Folding of Mistic

• Unlike the secondary structure determination,
  long-range restraints are necessary to determine
  the fold of the protein
• The monocysteine mutant library described in the
  topology assay was used to incorporate
  site-directed spin labels within Mistic that
  produce distance-dependent line- broadening
  perturbations in the NMR spectra that could be
  translated into distances for structure
  determination
• The signal changes observed for the five
  spin-labeled samples were transformed into 197
  long-range upper-distance and 290 lower- distance
  restraints

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Results

• After collecting all the NOE data, angle
  restraints, spin labeling restraints and
  a-helical hydrogen bond restraints, the final
  structure calculation resulted in
• 1. 573 NOE distance restraints
• 2. 346 angle restraints from chemical shifts
  and NOEs
• 3. 478 distance restraints from spin-label
  experiments

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3-D Structure of Mistic

• The bundle of 10 conformers with the lowest
  target function is used to represent the
  three-dimensional NMR structure.
• The loop connecting a2 and a3, as well as the C
  terminus of Mistic, are more mobile. (This proves
  to be important further into the experiment)

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• All helices except a2 are slightly shorter than
  expected for a bilayer- traversing helix
• This is likely due to partial unraveling of the
  ends of the helices in the detergent micelle
  environment, especially at the N and C termini
  (a1 and a4) allows Mistic to adapt to
  the lipid environment
• Helix a2 has a kink

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Surprising Structure of Mistic

• Mistic appears to have hydrophilic surface for an
  IM protein even though it is assembled internally
  with a typical hydrophobic core.
• Given the membrane-traversing topology
  demonstrated by the MPB labeling experiment this
  is an unusual surface property.

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Confirming The Unusual Hydrophilic Surface

• NOEs between Mistic and its solubilizing LDAO
  detergent micelle were measured and assigned.
• When sites with NOE signals are mapped to the
  surface of the Mistic structure, a concentric
  ring of detergent interactions around the helical
  bundle is observed, as expected for a
  membrane-integrated protein.
• Results
• Mistic is embedded within the LDAO micelle.

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Variable Conformation

• Mistic might be exploited to target another
  protein to the bacterial membrane, when fused to
  Mistics C terminus, such that it too could
  readily fold into its native, lipid bilayer
  inserted conformation.
• Mistic-assisted expression of three topologically
  and structurally distinct classes of eukaryotic
  IM proteins were tested
• 1. voltage-gated K channels
• 2. receptor serine kinases of the transforming
  growth factor-ß (TGF-b) superfamily
• 3. G-protein coupled receptors (GPCRs)
• Result
• In 15 of the 22 tested constructs the desired
  product could be isolated from the membrane
  fraction of recombinant bacteria at yields
  exceeding 1 mg per liter of culture.

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• Figure B
• The Mistic-fused protein is shown on the left
  (open arrow)
• The final product after removal of Mistic by
  thrombin digestion is on the right (solid arrow).

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Mistic Produces High Yields of IM Proteins

• The identity of the resulting bands are
  determined by N-terminal sequencing
• In addition, aKv1.1 was extracted and purified in
  LDAO to verify that the protein resembled its
  native conformation. Gel-filtration showed the
  structure is a tetramer.
• Results
• There exists a high propensity for this system
  to produce IM proteins fully folded in their
  native conformations

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Mutational Disruption of Mistics Structure and
Function

• Mutations at three potentially structurally
  disruptive sites within the core of the protein
  W13, Q36, and M75.
• Results show that Mistics structure is essential
  to its ability to chaperone cargo proteins to the
  bacterial lipid bilayer.
• For example
• The single mutation of a core methionine (Met75)
  to alanine destabilized Mistics structure such
  that it partitioned between the membrane and the
  cytoplasm. This resulted in no protein expression
  when fused to aKv1.1

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W Tryptophan M Methionine Q Glutamine
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Conclusion

• All available data suggest that Mistic must
  autonomously associate with the bacterial
  membrane and that this property alone accounts
  for its high efficiency in chaperoning the
  production and integration of downstream cargo
  proteins.
• Conformational flexibility, such as rotation of
  the four helices about their helical axes or even
  partial unraveling of the helical bundle, may
  allow Mistic to adapt to lipid environments.
• Mistic retains an unexpectedly hydrophilic
  surface for an IM protein even though it is
  assembled internally with a typical hydrophobic
  core.
• Mistics ability to help produce high yields of
  eukaryotic integral membrane proteins has and
  will enhance research in that area greatly.

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References

1. Roosild, Tarmo P., Jason Greenwald, Mark
   Vega,Samantha Castronovo, Roland Riek, and
   Senyon Choe. "NMR Structure of Mistic, a
   Membrane-Integrating Protein for Membrane
   Protein Expression." Science. 25 Feb. 2005.
   Web. ltwww.sciencemag.orggt.