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PDBsum entry 1jfq
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Immune system
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PDB id
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1jfq
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural analysis of mutants of high-Affinity and low-Affinity p-Azophenylarsonate-Specific antibodies generated by alanine scanning of heavy chain complementarity-Determining region 2.
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Authors
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B.Parhami-Seren,
M.Viswanathan,
R.K.Strong,
M.N.Margolies.
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Ref.
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J Immunol, 2001,
167,
5129-5135.
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PubMed id
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Abstract
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Alanine scanning was used to determine the affinity contributions of 10 side
chain amino acids (residues at position 50-60 inclusive) of H chain
complementarity-determining region 2 (HCDR2) of the somatically mutated
high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was
expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains
to also assess the contribution of L chain mutations to affinity. Combined data
from fluorescence quenching, direct binding, inhibition, and capture assays
indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain
results in significant loss of binding with both mutated (36-71L) or unmutated
(36-65L) L chain, although the decrease was more pronounced when unmutated L
chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab
affinity when expressed with 36-71 L chain. However, in the context of unmutated
L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs
containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited
4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead
on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and
expressed with unmutated L chain as Fab in bacteria, these mutants exhibited
affinities similar to or slightly higher than the wild-type 36-65. These
findings indicate an important role of certain HCDR2 side chain residues on Ab
affinity and the constraints imposed by L chain mutations in maintaining Ag
binding.
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