Gustavsson, Martin

Ha, Kim H.

Traaseth, Nathaniel J.

Veglia, Gianluigi

Verardi, Raffaello

Elsevier - Publisher Connector 

Tuesday, March 3, 2009 431aoligosaccharide head group for hydrolytic cleavage by the hydrolase, HexA.
Mutations in GM2AP or HexA lead to an accumulation of GM2 in the lyso-
somes, causing lysosomal storage diseases such as Tay Sachs or the AB variant
of Sandhoff’s disease. Our lab is utilizing site directed spin-labeling (SDSL)
with electron paramagnetic resonance (EPR) spectroscopy to probe the confor-
mational changes and the membrane bound orientation of GM2AP. This pro-
tein contains eight naturally occurring cysteine (CYS) residues involved in
four disulfide bonds. With site directed mutagenesis, a ninth CYS residue is
introduced as the reporter site for spin labeling . A series of 10 single CYS mu-
tants have been generated. To validate the EPR results, the mass spectrometry
protocol described here was developed to characterize spin-labeled GM2AP
samples. For mass spectrometry measurements, either biotin-linked maleimide
(BM) or 4-maleimide tempo (4MT) were used to modify and trap available
CYS residues in a thioesterbond. The remaining eight native CYS residues,
which are disulfide bonded, are then reduced and modified with iodoacetamide.
Samples were analyzed by high performance nano-liquid chromatography
electrospray ionizaton Fourier-transform ion cyclotron resonance mass spec-
trometry (FT-ICR MS) equipped with a 14.5 T magnet. FT-ICR spectra of
enzymatically digested BM-labeled or 4MT-labeled GM2AP were utilized to
determine to which CYS residue is modified with the maleimide moeity. Those
sites labeled with acetamide are inferred to have been disulfide bonded. The
fragment that contains the maleimide moeity tells us, which CYS residue
(and how many CYS residues) is accessible for reaction with the spin label
for EPR studies.
2220-Pos Board B190
Purification And Reconstitution Of The Connexin43 Carboxyl Terminus
Attached To The 4Th Transmembrane Domain In Detergent Micelles
Rosslyn Grosely, Admir Kellezi, Fabien Kieken, Gloria E.O. Borgstahl,
Paul L. Sorgen.
UNMC, Omaha, NE, USA.
In recent years, reports have identified that many eukaryotic proteins contain
disordered regions spanning greater than 30 consecutive residues in length.
In particular, a number of these intrinsically disordered regions occur in the cy-
toplasmic segments of plasma membrane proteins. These intrinsically disor-
dered regions play important roles in cell signaling events, as they are sites
for protein-protein interactions and phosphorylation. Unfortunately, in many
crystallographic studies of membrane proteins, these domains are removed be-
cause they hinder the crystallization process. Therefore, a purification proce-
dure was developed to enable the biophysical and structural characterization
of these intrinsically disordered regions while still associated with the lipid en-
vironment. The carboxyl-terminal domain from the gap junction protein con-
nexin43 attached to the 4th transmembrane domain (TM4-Cx43CT) was used
as a model system (residues G178-I382). The purification was optimized for
structural analysis by nuclear magnetic resonance (NMR) because this method
is well suited for small membrane proteins and proteins that lack a well-struc-
tured three-dimensional fold. The TM4-Cx43CT was purified to homogeneity
with a yield of ~6 mg per liter from C41(DE3) bacterial cells, was reconstituted
in the anionic detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-
(1-glycerol)], and circular dichroism and NMR were used to demonstrate
that the TM4-Cx43CT was properly folded into a functional conformation by
its ability to form a-helical structure and associate with a known binding part-
ner, the c-Src SH3 domain, respectively.
2221-Pos Board B191
A Refinement Protocol to Define the Structure, Topology and Depth of
Insertion of Membrane Proteins using Hybrid Solution/Solid-state NMR
Restraints
Lei Shi, Nathaniel J. Traaseth, Raffaello Verardi, Gianluigi Veglia.
University of Minnesota, Minneapolis, MN, USA.
To fully describe the folding space and ultimately the biological function of
membrane proteins (MPs), it is necessary to determine their interactions with
lipid membranes. This intrinsic property of MPs referring to as structural topol-
ogy is not possible to resolve using x-ray crystallography or solution NMR.
Here, we present a hybrid solution and solid-state NMR approach for the simul-
taneous determination of the structure, topology, and depth of insertion of MPs.
Distance and angle restraints obtained from solution NMR of MPs solubilized
in detergent micelles are combined with backbone orientational restraints (15N
chemical shift anisotropy and 15N-1H dipolar couplings) derived from solid-
state NMR in orientated lipid bilayers (PISEMA) into a hybrid objective func-
tion. In addition, a supplementary energy term, Ez (insertion depth potential), is
used to ensure the correct positioning of helical MPs domains with respect to
a virtual membrane. The hybrid objective function is optimized using a two-
stage simulated annealing protocol and is implemented into XPLOR-NIH soft-
ware for general use. To validate the hybrid method, the effects of chemicalshift tensor orientations, principal tensor values, and dipolar constant magni-
tudes on the structural ensemble are determined.
The hybrid method is applied to monomeric and pentameric PLN (phospholam-
ban), a integral MP that regulates sarco(endo)plasmic reticulum Ca-ATPase
(SERCA) function in cardiac muscle. The hybrid conformational ensemble de-
fines the structure, topology and depth of insertion of PLN in lipid bilayer si-
multaneously. This ensemble is compared with solution NMR structures in
DPC micelle obtained using conventional solution NMR data (NOEs, J-
couplings) and residual dipolar coupling as orientational restraints.
This research was supported by NIH Grants GM64742 and K02HL080081 to
G.V. and we also thank University of Minnesota, Supercomputing Institute
(MSI) for providing the computing resource.
2222-Pos Board B192
Solution and Solid-State NMR Analysis of Phosphorylated and Pseudo-
Phosphorylated Phospholamban
Raffaello Verardi, Nathaniel J. Traaseth, Martin Gustavsson, Kim H. Ha,
Gianluigi Veglia.
University of Minnesota, Minneapolis, MN, USA.
Phospholamban (PLN) is a 52 residue transmembrane protein that regulates the
Ca2þ ATPase (SERCA) of muscle cells. At low mM Ca2þ concentration, PLN
binds to SERCA and decreases the rate of uptake of Ca2þ into the sarcoplasmic
reticulum (SR) thereby promoting muscle relaxation. This inhibitory activity is
reversed by phosphorylation of PLN at Serine-16 or Threonine-17 in response
to ß-adrenergic stimulation. Phosphorylation of PLN is associated with in-
creased cardiac contractility of muscle cells due to the larger Ca2þ load into
the SR.
PLN has become important as a therapeutic target in fighting heart failure,
a complex disease associated with impaired cardiac contractility. It has been
shown that delivering of phospho-mimiking PLN (Ser-16/ Glu substitution)
to failing mice cardiomyocytes significantly improves contractility.
Although functional effects of PLN phosphorylation have been extensively
studied, the mechanistic details of how phosphorylated and pseudo-phosphor-
ylated (S16E) PLN interacts with SERCA to reverse inhibition are still unclear.
In here we present data on the structural characterization of Ser-16 phosphor-
ylated and S16E monomeric and pentameric phospholamban (AFA-PLN and
WT-PLN) in the presence and absence of SERCA as probed by solution and
solid-state NMR spectroscopy in detergent micelles and oriented lipid bilayers.
For solid-state NMR, SERCA and PLN were reconstituted in planar lipid bila-
yers and uniaxially aligned on glass plates. Residue-specific information as
well as topology of PLN monomer and pentamer was determined by PISEMA
experiments.
2223-Pos Board B193
Hybrid Solution and Solid-State NMR Analysis of SERCA/Phospholam-
ban Interactions in lipid membranes: From Structural Dynamics to Func-
tion
Gianluigi Veglia, Nathaniel J. Traaseth, Raffaello Verardi, Lei Shi, Kim Ha.
University of Minnesota, Minneapolis, MN, USA.
Phospholamban (PLN) is a sarcoplasmic reticulum (SR) integral membrane
protein that regulates calcium translocation in cardiac muscle. Upon interaction
with SERCA (the SR calcium ATPase). PLN decreases the rate of calcium up-
take, reducing the apparent affinity of the enzyme for Ca2þ ions. This process is
reversed by adrenergic stimulation of protein kinase A, which phosphorylates
PLN at Ser16, re-starting the muscle contraction cycle. Here, we present the hy-
brid solution and solid-state NMR structural analysis of PLN in its pentameric
(storage), monomeric (active), and SERCA-bound forms in lipid membranes.
This knowledge about the structural dynamics PLN under these different stages
is used to steer the extent of PLN control on SERCA activity. These findings lay
the groundwork for the rational design of PLN loss-of-function mutants to be
used in gene therapy.
2224-Pos Board B194
Topology of Phospholamban when Bound to Ca25ATPase by Solid-State
NMR
Nathaniel Traaseth, Raffaello Verardi, Lei Shi, Gianluigi Veglia.
University of Minnesota, Minneapolis, MN, USA.
Calcium cycling in muscle cells drives the relaxation and contraction of both
skeletal and heart tissue. The sarcoplasmic reticulum (SR) Ca2þ-ATPase is
central in the relaxation of the heart, accounting for ~70% of calcium seques-
tration. Phospholamban (PLN) is a small integral membrane protein regulator
of Ca2þ-ATPase. Its inhibition of the enzyme is shown in calcium dependence
ATPase activity curves, resulting in decreased ATPase affinity for Ca2þ. While
there have been several successful attempts to gain structural knowledge of the
complex between PLN and Ca2þ-ATPase, no high-resolution structure exists.


Solution and Solid-State NMR Analysis of Phosphorylated and Pseudo-Phosphorylated Phospholamban 

file:///data/core-remote/dit/data/elsevier/pdf/3e6/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUwODAyNDMwMg==.pdf

Solution and Solid-State NMR Analysis of Phosphorylated and Pseudo-Phosphorylated Phospholamban

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector