Transcript NMR Structure of Mistic, a Membrane

NMR Structure of Mistic, a
Membrane-Integrating Protein
for Membrane Protein
Expression
By: Niloufar Safvati
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
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.
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
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)
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
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 membraneembedded in Mistic’s native conformation.
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
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.
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 ahelical secondary structure
•Values smaller than -1.5 ppm are indicative of ßsheet secondary structure.
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.
Transverse Relaxation Optimized
Spectroscopy (TROSY)
Fernandex and Wider, Current Opinion in Structural Biology 2003, 13:570-580
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.
Folding of Mistic
 Unlike the secondary structure determination, longrange 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 distancedependent 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
Results

After collecting all the NOE data, angle
restraints, spin labeling restraints and α-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
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 α2 and α3, as well as the C terminus
of Mistic, are more mobile. (This proves to be important
further into the experiment)
 All helices except α2 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 (α1 and α4)
allows Mistic to
adapt to the lipid environment
 Helix α2 has a kink
Surprising Structure of Mistic
Quic kTime™ and a
dec ompres s or
are needed to s ee this pic tur e.
•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.
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.
Variable Conformation
 Mistic might be exploited to target another protein to the
bacterial membrane, when fused to Mistic’s 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.
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).
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
Mutational Disruption of Mistic’s
Structure and Function
• Mutations at three potentially structurally disruptive sites
within the core of the protein: W13, Q36, and M75.
• Results show that Mistic’s 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 Mistic’s structure such that it
partitioned between the membrane and the cytoplasm. This
resulted in no protein expression when fused to aKv1.1
W: Tryptophan
M: Methionine
Q: Glutamine
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.
 Mistic’s ability to help produce high yields of eukaryotic
integral membrane proteins has and will enhance research
in that area greatly.
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. <www.sciencemag.org>.