title: Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of transcription factor GCN4.
abstract: Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast transcription factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.
authors.affiliation: Biochemisches Institut der Universität Zürich, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Weber-Bornhauser
authors.forename: J
authors.initials: J
authors.lastname: Eggenberger
authors.forename: I
authors.initials: I
authors.lastname: Jelesarov
authors.forename: A
authors.initials: A
authors.lastname: Bernard
authors.forename: C
authors.initials: C
authors.lastname: Berger
authors.forename: H R
authors.initials: HR
authors.lastname: Bosshard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D002151:Calorimetry; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005454:Fluorescence Polarization; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016350:Leucine Zippers / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D000939:Epitopes; D005656:Fungal Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; D014157:Transcription Factors; C055966:immunoglobulin Fv; C114805:scFv fragment SW1; D011494:Protein Kinases