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PDBsum entry 3eot
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Immune system
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PDB id
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3eot
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References listed in PDB file
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Key reference
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Title
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An antibody loop replacement design feasibility study and a loop-Swapped dimer structure.
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Authors
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L.A.Clark,
P.A.Boriack-Sjodin,
E.Day,
J.Eldredge,
C.Fitch,
M.Jarpe,
S.Miller,
Y.Li,
K.Simon,
H.W.Van vlijmen.
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Ref.
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Protein Eng Des Sel, 2009,
22,
93.
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PubMed id
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Abstract
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A design approach was taken to investigate the feasibility of replacing single
complementarity determining region (CDR) antibody loops. This approach may
complement simpler mutation-based strategies for rational antibody design by
expanding conformation space. Enormous crystal structure diversity is available,
making CDR loops logical targets for structure-based design. A detailed analysis
for the L1 loop shows that each loop length takes a distinct conformation,
thereby allowing control on a length scale beyond that accessible to simple
mutations. The L1 loop in the anti-VLA1 antibody was replaced with the L2 loop
residues longer in an attempt to add an additional hydrogen bond and fill space
on the antibody-antigen interface. The designs expressed well, but failed to
improve affinity. In an effort to learn more, one design was crystallized and
data were collected at 1.9 A resolution. The designed L1 loop takes the
qualitatively desired conformation; confirming that loop replacement by design
is feasible. The crystal structure also shows that the outermost loop (residues
Leu51-Ser68) is domain swapped with another monomer. Tryptophan fluorescence
measurements were used to monitor unfolding as a function of temperature and
indicate that the loop involved in domain swapping does not unfold below 60
degrees C. The domain-swapping is not directly responsible for the affinity
loss, but is likely a side-effect of the structural instability which may
contribute to affinity loss. A second round of design was successful in
eliminating the dimerization through mutation of a residue (Leu51Ser) at the
joint of the domain-swapped loop.
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