Beck-Sickinger, Annette G.

Chollet, Constance

Huster, Daniel

Scheidt, Holger A.

Thomas, Lars

Vortmeier, Gerrit

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Solid-State NMR Studies on the Membrane Bound Acylated Peptide Hormone Ghrelin 

264a Monday, February 27, 2012activated when the natural ligand PDGF binds simultaneously to the ectodo-
mains of two receptors, resulting in spatial proximity and subsequent
cross-activation of the cytosolic kinase domains1). The receptor can also be
activated independently of the natural ligand by binding of the small E5 oncopro-
tein from bovine papillomavirus2). E5 is a small membrane protein of only 44
amino acids and it is thought to manipulate the function of the receptor by
specific helix-helix-contacts to the transmembrane of the receptor which then
result in sustained receptor activation and cell transformation.
To elucidate the helix-helix-interactions in receptor complex, we investigate
the structure and membrane alignment of both proteins, first for each protein
and later in the heterotetrameric four-helix-bundle. For the structural analysis
we combined circular dichroism (CD) spectroscopy and liquid-state NMR to
determine the secondary structure. Oriented CD and solid-state NMR were
used to resolve the orientation of both proteins in their native environment.
Therefore the E5 protein and the transmembrane domain of the receptor
(PDGFRTMD) were 15N-isotope labelled by bacterial protein expression
and reconstituted in detergent micelles and in oriented lipid bilayers. By
CD and liquid-state NMR we found that both proteins are predominantly a-
helical in detergent micelles and in lipid bilayers3,4). Furthermore, high res-
olution NMR measurements showed that PDGFRTMD forms a left-handed
coiled-coil structure. A complementary OCD and solid-state NMR analysis
of E5 and PDGFRTMD reconstituted in different lipid bilayers showed that
the orientation of both proteins depends on the bilayer thickness, where
both proteins were more tilted in thin membranes and less tilted in thick
membranes.
1335-Pos Board B105
Structure and Dynamics of Membrane-Associated Hepatitis C Virus
Protein P7 by NMR Spectroscopy
Gabriel A. Cook, Lindsay Dawson, Stanley J. Opella.
University of California San Diego, La Jolla, CA, USA.
P7 is a 63-residue viroporin encoded by human hepatitis C virus (HCV). It has
ion channel activity and plays essential roles in viral proliferation. The NMR
results show that the protein consists of two transmembrane spanning regions
connected by a cytoplasmic loop. The solution NMR structural data that has
been acquired includes: hydrogen/deuterium exchange, paramagnetic relaxa-
tion enhancement, residual dipolar couplings, and bicelle ‘q-titration.’ These
data demonstrate that the protein has a range of dynamic properties, and in-
terestingly two segments in each of the membrane spanning helices display
local motions, possibly related to function. Oriented Sample (OS) solid-
state NMR spectra of p7 in aligned phospholipid bilayers provided the tilt an-
gles of the helical segments, 25 and 10 to the membrane normal. Recent
magic angle spinning (MAS) solid-state NMR data of p7 in liposomes are be-
ing used to determine the three-dimensional structure. It has been shown that
known channel-blocking compounds inhibit the ion channel activity. This and
the fact that knockout models of this protein inhibit infectivity of the virus in-
dicate it may be an ideal target for future drugs. There are six major genotypes
and over 100 relevant subtypes. Our current studies aim to investigate the
structure and dynamic differences among the genotypes. In addition to study-
ing different genotypes, specific mutations to the HCV J4 genotype were
made in order to modulate the dynamics of the protein and obtain additional
secondary structural information. These studies have the potential to identify
the reasons why different genotypes have varying activity and drug binding
affinities.
1336-Pos Board B106
Solid-State NMR Studies on the Membrane Bound Acylated Peptide
Hormone Ghrelin
Gerrit Vortmeier, Constance Chollet, Holger A. Scheidt, Lars Thomas,
Annette G. Beck-Sickinger, Daniel Huster.
University of Leipzig, Leipzig, Germany.
Ghrelin is a 28-amino acid peptide, which is known to play a key role in the
regulation of food intake and body weight. Ghrelin is the only known hor-
mone that carries an octanoyl lipid modification at residue Ser 3. Here, we
have studied the structure and dynamics of the molecule bound to DMPC/
DMPS (80/20) membranes by solid-state NMR spectroscopy. We have syn-
thesized several Ghrelin peptides with varying 13C/15N containing amino
acids covering 10 out of the 28 residues in the sequence. 13C NMR spectra
recorded under magic-angle spinning conditions provide backbone torsion an-
gles for the membrane-associated peptide. Further, the molecular dynamics of
Ghrelin was studied using motional averaged dipolar coupling measurements.
Overall, the data indicates a rather flexible peptide conformation on the mem-brane surface. We have further studied the conformation and membrane inser-
tion of the octanoyl lipid modification of Ghrelin using 2H NMR. Ghrelin
binding leads to a small alteration in the order parameter profile of the host
membrane. Further, we have also measured the 2H NMR spectrum of a Ghre-
lin molecule with a deuterated octanoyl chain. This spectrum showed clear in-
dications of a membrane inserted lipid chain, however, the chain order
parameters of the lipid modification are smaller translating into a somewhat
shorter chain length compared to the host membrane. Experimentally, we de-
termined a membrane binding energy of ~40 kJ/mol for Ghrelin, which cannot
solely be explained by the insertion of hydrophobic amino acids and the oc-
tanoyl chain. In addition, electrostatic forces play a role arising from the 7
positively charged residues in the peptide sequence. In the course of action,
Ghrelin binds to the GHS receptor, so membrane binding might be an impor-
tant step increasing the Ghrelin concentration at the membrane prior to bind-
ing to its receptor.
1337-Pos Board B107
Towards the Backbone Structure Determination of the Membrane Pro-
tein, LspA, from Mycobacterium Tuberculosis using Solid State NMR
E. Vindana Ekanayake1,2, D.Q. Ni1, Timothy A. Cross1,2*.
1The Florida State University, Tallahassee, FL, USA, 2National High
Magnetic Field Laboratory, Tallahassee, FL, USA.
Lipoprotein Signal Peptidase A (LspA) is an essential enzyme in one of the
three distinct steps in lipoprotein maturation. After targeting, the signal peptide
of a prolipoprotein, the peptide is cleaved by LspA to result in the mature
lipoprotein.
In Mycobacterium tuberculosis (Mtb), LspA is encoded by the gene RV1539
resulting in a 202 amino acid residue peptide with a molecular weight is 21
kDa that is predicted to possess four transmembrane helices. It has been rec-
ognized in vivo that LspA is an essential protein for Mtb virulence and that
it may also contribute to antibiotic resistance, as a result LspA has been
identified as a potential drug target for the treatment of tuberculosis. Globo-
mycin, a cyclic-peptide is known to inhibit LspA in a non-competitive
manner.
Solid-state NMR uniformly aligned samples is being used to determine the
structure of the protein backbone. Anisotropic 15N-1H dipolar couplings and
15N chemical shifts are obtained from separated local field NMR spectra of
the protein in aligned bilayer preparations. The resulting orientational re-
straints can be used to generate a high resolution structure as well as a de-
termination of how the helices are oriented with respect to their
environment.
In this study, LspA is reconstituted into synthetic lipids (DMPC/DMPG and
POPC/POPG) to mimic the membrane environment and it is aligned uniaxially
either mechanically (using glass slides) or magnetically (using bicelles).
1338-Pos Board B108
Initial Structural Studies of Mtb Membrane Protein ‘‘CrgA’’ in Mem-
brane Mimetic Environment
Nabanita Das1, Ivan Hung2, Huajun Qin1, Timothy A. Cross1.
1Florida State University, Tallahassee, FL, USA, 2National High Magnetic
Field Laboratory, Tallahassee, FL, USA.
Here we present initial NMR Spectroscopic studies of CrgA, one of the impor-
tant putative inner membrane proteins in Mycobacterium tuberculosis (Mtb).
CrgA helps in the cell division of Mtb contains 2 transmembrane helices
(TM), 93 amino acid and is an important drug target. Our studies show that,
CrgA interacts with FtsZ (involves in Zring formation during cell division)
and Rv0008c (another putative membrane protein) in Mtb during the cell divi-
sion. For NMR structural studies 15N, 13C uniform and amino acid specific iso-
tope labeled CrgA reconstituted in POPC-POPG (4:1 mol/mol) liposomes was
used for planar lipid bilayer oriented on glass slides and Magic Angle Spinning
(MAS) correlation spectroscopy. The key behind these studies is to determine
CrgA structure in membrane mimetic lipid bilayer environment. For oriented
glass slide samples, 2D PISEMA experiments were performed which correlates
1H-15N dipolar coupling with the 15N chemical shift from the backbone reso-
nances of 15N labeled protein in lipid bilayer. Each measured frequency reflects
the orientation of the peptide plane of the protein w.r.t to the bilayer normal.
Similarly the tilt angles of various parts of the protein (TM, N and C terminal)
can also be obtained which is very useful for the structure calculation process.
Moreover MAS experiments can measure distances between side chains of var-
ious residues. Altogether the aim of this research is to characterize the 3D back-
bone as well as the side chain structures of CrgA in native membrane like
environment.


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Solid-State NMR Studies on the Membrane Bound Acylated Peptide Hormone Ghrelin

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