1jfq: Difference between revisions

Revision as of 14:22, 21 February 2008

ANTIGEN-BINDING FRAGMENT OF THE MURINE ANTI-PHENYLARSONATE ANTIBODY 36-71, "FAB 36-71"

OverviewOverview

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.

About this StructureAbout this Structure

1JFQ is a Protein complex structure of sequences from Mus musculus. Full crystallographic information is available from OCA.

ReferenceReference

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., Parhami-Seren B, Viswanathan M, Strong RK, Margolies MN, J Immunol. 2001 Nov 1;167(9):5129-35. PMID:11673524

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 ==Overview==
-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.
+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.
 ==About this Structure==
@@ Line 13: / Line 13: @@
 [[Category: Mus musculus]]
 [[Category: Protein complex]]
-[[Category: Margolies, M.N.]]
+[[Category: Margolies, M N.]]
 [[Category: Parhami-Seren, B.]]
-[[Category: Strong, R.K.]]
+[[Category: Strong, R K.]]
 [[Category: Viswanathan, M.]]
 [[Category: immunoglobulin]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri Feb 15 16:07:12 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:22:08 2008''