Kanagawa, Osami

Ogawa, Naoki

Sasaki, Yuji C.

Elsevier - Publisher Connector 

Molecular un-stability of peptide-MHC complex and activation of T cell 

680a Wednesday, March 4, 2009of detergent micelle resonances, a problem overcome by the selective exci-
tation approach.
3507-Pos Board B554
An Important Double Bond: Effects of 22:5n-6 vs. 22:6n-3 on Visual Signal
Transduction
Drake C. Mitchell1, Shui-Lin Niu2.
1Portland State University, Portland, OR, USA, 2NIH/NIAAA, Bethesda,
MD, USA.
In a normal, healthy retinal rod outer segment 40% to 50% of the phospholipid
acyl chains consist of docosahexaenoic acid (DHA, 22:6n-3). Diets that are de-
ficient in n-3, oru-3, fatty acids lead to the replacement of 22:6n-3 with 22:5n6.
Dietary n-3 deficiency leads to a spectrum of developmental disorders associ-
ated with learning, memory, intelligence, and visual function. We examined
rhodopsin, transducin (Gt) and phosphodiesterase (PDE) function and acyl
chain packing in large unilamellar proteoliposomes consisting of phosphatidyl-
cholines with sn-1 ¼ 18:0, and sn-2 ¼ 22:6n-3, 22:5n-6 or 22:5n-3. Rhodopsin
activation and binding to Gt was assayed with steady-state and time-resolved
UV/vis spectroscopy, acyl chain packing was assessed via time-resolved fluo-
rescence of diphenylhexatriene (DPH) and PDE activity was determined from
the change in pH due to hydrolysis of cyclic GMP. The motion of DPH in the
membrane was slower in 22:5n-6 than in 22:6n-3 and overall acyl chain pack-
ing was more constrained. The most significant structural difference between
the 22:5n-6 containing bilayer and bilayers containing both n-3 polyunsaturates
was in the bilayer mid plane where 22:5n-6 produced much higher DPH orien-
tational order. At physiological temperature the formation of both the active
metarhodopsin II conformation (MII) and the MII-Gt complex was much
slower in 18:0,22:5n-6 PC than in 18:0,22:6n-3 PC and the equilibrium amount
of MII formed was 50% higher in 18:0,22:6n-3 PC. In 18:0,22:5n-6 PC PDE
activity at a physiologically relevant level of rhodopsin activation is only about
60% of that observed in either 18:0,22:6n-3 PC or 18:0,22:5n-3 PC. Taken to-
gether, these results demonstrate that the subtle change in bond configuration
from 22:6n-3 to 22:5n-6 produces more structured acyl chain packing in the bi-
layer midplane, leading to delayed and reduced MII-Gt interaction and PDE
function.
3508-Pos Board B555
Dynamics of the Internal Water Molecules in Squid Rhodopsin
Eduardo Jardo´n-Valadez, Ana-Nicoleta Bondar, Douglas J. Tobias.
UCI-Irvine, Irvine, CA, USA.
G-protein coupled receptors (GPCRs) are major parmaceutical targets because
of their key role in a diverse array of physiological functions. The visual rho-
dopsin is the prototype for the family A of GPCRs. Upon photoisomerization of
the covalently-bound retinal chromophore, visual rhodopsins undergo a large-
scale conformational change that prepares the receptor for a productive inter-
action with the G protein. The mechanism by which the local perturbation of
the retinal cis-trans isomerization is transmitted throughout the protein is not
well understood. The recently reported crystal structure of squid rhodopsin
(M. Murakami and T. Kouyama, Nature 453, 363, 2008) displays new features
that may provide additional insight into the mechanism of the signal transduc-
tion in GPCRs. It has been suggested, based on the location of water molecules
in the interhelical region extending from the retinal towards the cytoplasmic
side, that a water-mediated hydrogen-bond network may play a role in the ac-
tivation process. As a first step towards understanding the role of water in rho-
dopsin function, we have performed a molecular dynamics simulation of squid
rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules.
Here we report results from the simulation that show that the water molecules
present in the crystal structure participate in favorable interactions with side
chains in the interhelical region, and form a persistent hydrogen-bond network
in connecting Tyr315 to Trp274 via Asp80. We also present preliminary results
from a simulation study of the changes in the structure and dynamics of the hy-
drogen-bond network that accompany the photoisomerization of the retinal
chromophore.
3509-Pos Board B556
Single-Molecule Photoactivation and Localization of Signaling Complexes
in T cells
Chih-Jung Hsu.
University of Pennsylvania, Philadelphia, PA, USA.
In T cells, membrane receptor and ligand engagement initiates a signaling cas-
cade. Ligand binding is relayed through specific membrane receptors, protein
tyrosine kinases, and critical adaptors to regulate downstream activation of
transcription factors, cytokine production and cell proliferation. The membrane
segregation and relocalization of these signaling components display highly dy-
namic protein networks in response to distinct stimulations. However, the
membrane-protein and protein-protein interactions involved in the formationof signaling complexes are still poorly understood. We are currently using
a high-resolution imaging technique to visualize the early stage of signal trans-
duction events at the single-molecule level with photoactivatable or photocon-
vertable fluorescent markers. The photoactivation and localization of T-cell
signaling components allow measurements of molecular spatial and temporal
correlation in response to different stimulations. With dual-color application,
molecular correlation functions will enhance the understanding of the pro-
tein-protein interactions in signaling complexes.
3510-Pos Board B557
Molecular un-stability of peptide-MHC complex and activation of T cell
Naoki Ogawa1, Yuji C. Sasaki2, Osami Kanagawa1.
1Research Center for Allergy and Immunology, RIKEN, Yokohama, Japan,
2Biomedical Group, SPring-8/JASRI (JST/CREST Sasaki-team), Sayou-gun,
Japan.
CD4þ T cell responses require the recognition of specific peptide-MHC com-
plexes displayed by APC. It is important to determine how Ag presentations
affect the ensuing T cell response. Imunization of B10.BR mice with immuno-
dominant peptide 48-61 of Hen egg lysozyme elicit two different types of T cell
responses. First type of T cell (type A termed by Unanue et. al) respond to APC
pulsed with either peptide or whole HEL protein. Second type of T cell (termed
type B) respond to APC incubated with peptide but showed no response to APC
with whole protein. Some of the type B T cell clone exhibit unusual response to
the variant of 48-61 peptide and responded better to poor MHC binding peptide
that to strong MHC binding peptide. In contrast, reactivity of the type A of
T cell clones correlate well with the affinity of the peptide to the MHC mole-
cules. Since weak MHC binding peptides form unstable complex, we hypoth-
esize that T cells, like type B T cell, respond well to unstable MHC peptide
complex by interacting with one of multiple transitional conformations. To
test this hypothesis, we observed the movement of peptide/MHC complex at
the single molecular level by using diffracted X-ray tracking (DXT) method.
It was found that movement of the low affinity peptide MHC complex was
greater than that of high affinity peptide MHC complex. However, the differ-
ence between two complexes was mainly due to the overall movement of the
molecule rather than the different movement of the peptides in the MHC
groove. Thus, peptide bound to the MHC greatly influence the movement of
the whole MHC peptide complex and this movement strongly affect the recog-
nition by T cell.
3511-Pos Board B558
The Influence Of Plasma Membrane Order On TCR Signalling
Ben de Wet, Brid Cronin, Mark Wallace, Thomas Harder.
University of Oxford, Oxford, United Kingdom.
The signalling events that follow T-cell receptor triggering are mediated by
multi-molecular complexes consisting of both membrane-associated and cyto-
solic proteins. Formation of these complexes is driven by a network of protein-
protein and protein-lipid interactions. We study how the selective partitioning
of signalling components into separate ordered/raft or disordered phases of the
plasma membrane controls the assembly and activity of these signalling com-
plexes.
Previous work from our laboratory shows that T cell plasma membrane do-
mains engaged in TCR signalling specifically undergo condensation, and that
treatment of Jurkat T-cells with polyunsaturated fatty acids specifically disrupts
condensed membrane rafts at TCR activation sites. We have also shown that
disruption of membrane order by 7-ketocholesterol leads to reduced recruit-
ment of key TCR signalling components as measured biochemically and by
TIRF microscopy of fixed cells. We aim to understand the influence of this bio-
physical phenomenon on the formation and composition of TCR signalling pro-
tein/lipid complexes.
To this end, we are currently studying the effects of raft disruption by 7-keto-
cholesterol and polyunsaturated fatty acids on the kinetic behaviour of TCR
signalling components. We employ single molecule real time TIRF microscopy
of fluorescently labelled TCR signalling proteins as well as biochemical and
proteomic analysis of TCR activation domains upon raft disruption.
3512-Pos Board B559
Dissecting T cell receptor nanocluster signaling
Boryana N. Manz1,2, Bryan L. Jackson1, Rebecca S. Petit1, Michael
L. Dustin3, Jay T. Groves1,4.
1Department of Chemistry, UC Berkeley, Berkeley, CA, USA, 2Biophysics
Graduate Group, UC Berkeley, Berkeley, CA, USA, 3Molecular Pathogenesis
Program, Skirball Institute of Biomolecular Medicine, NYU School of
Medicine, New York, NY, USA, 4Howard Hughes Medical Institute, Chevy
Chase, MD, USA.
The early steps of T cell activation involve the spatial rearrangement of T cell
receptors (TCR) and associated molecules into small nanoclusters at the


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Molecular un-stability of peptide-MHC complex and activation of T cell

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