Signal and binding. II. Converting physico-chemical responses to macromolecule-ligand interactions into thermodynamic binding isotherms.
Physico-chemical titration techniques are the most commonly used methods in characterizing molecular interactions. These methods are mainly based on spectroscopic, calorimetric, hydrodynamic, etc., measurements. However, truly quantitative physico-chemical methods are absolutely based on the determination of the relationship between the measured signal and the total average degree of binding in order to obtain meaningful interaction parameters. The relationship between the observed physico-chemical signal of whatever nature and the degree of binding must be determined and not assumed, based on some ad hoc intuitive relationship/model, leading to determination of the true binding isotherm. The quantitative methods reviewed and discussed here allow an experimenter to rigorously determine the degree of binding and the free ligand concentration, i.e., they lead to the construction of the thermodynamic binding isotherm in a model-independent fashion from physico-chemical titration curves.
Interactions of the Escherichia coli DnaB-DnaC protein complex with nucleotide cofactors. 1. Allosteric conformational transitions of the complex.
Interactions of nucleotide cofactors with both protein components of the Escherichia coli DnaB helicase complex with the replication factor, the DnaC protein, have been examined using MANT-nucleotide analogues. At saturation, in all examined stationary complexes, including the binary, DnaB-DnaC, and tertiary, DnaB-DnaC-ssDNA, complexes, the helicase binds six cofactor molecules. Thus, protein-protein and protein-DNA interactions do not affect the maximum stoichiometry of the helicase-nucleotide interactions. The single-stranded DNA dramatically increases the ATP analogue affinity, while it has little effect on the affinity of the NDP analogues, indicating that stationary complexes reflect allosteric interactions between the DNA- and NTP-binding site prior to the cofactor hydrolysis step and subsequent to product release. In the binary complex, the DnaC protein diminishes the intrinsic affinity and increases the negative cooperativity in the cofactor binding to the helicase; an opposite effect of the protein on the cofactor-helicase interactions occurs in the tertiary complex. The DnaC protein retains its nucleotide binding capability in the binary and tertiary complexes with the helicase. Surprisingly, the DnaC protein-nucleotide interactions, in the binary and tertiary complexes, are characterized by positive cooperativity. The DnaC assembles on the helicase as a hexamer, which exists in two conformational states and undergoes an allosteric transition, induced by the cofactor. Cooperativity of the allosteric transition depends on the structure of the phosphate group of the nucleotide. The significance of the results for the DnaB-DnaC complex activities is discussed.
Subject index
Adoptive immunotherapy A1, A63, A63, A64, A64, A65, A66, A68, A69, A69, A70, A70, A71, A71, A72, A72, A73, A73, A74, A75, A76, A78, A79, A79, A7, A80, A80, A81, A81, A82, A84, A85, A86, A86, A88, A88, A89, A90, A90, A91, A92, A93, A93, A94, A95, A95, A96, A96, A97, A97, A98, A98, A99, A99, A102, A103, A104, A104, A166, A167, A169, A170, A174, A177, A179, A181, A182, A199, A204, A205, A21, A215, A220, A231, A231, A243, A251, A254, A25, A276, A287, A304, A307, A310, A315, A316, A316, A329, A331, A333, A357, A358, A369, A405, A441, A442, A458, A458, A459, A459, A461, A461, A463, A468, A468, A479, A492, A497, A497, A56, A58, A58, A59, A60, A60, A61, 1, 100, 101, 102, 103, 104, 106, 109, 111, 112, 113, 114, 115, 116, 117, 119, 120, 122, 123, 124, 125, 127, 128, 129, 13, 130, 131, 133, 134, 136, 139, 140, 141, 142, 146, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 163, 164, 165, 166, 167, 169, 173, 174, 175, 272, 273, 278, 280, 285, 290, 293, 297, 298, 324, 333, 335, 35, 353, 360, 379, 380, 400, 414, 418, 42, 454, 470, 493, 500, 504, 513, 515, 516, 542, 545, 549, 598, 599, 614, 675, 737, 739, 764, 765, 766, 767, 770, 771, 772, 780, 781, 801, 822, 830, 831, 89, 91, 92, 94, 95, 96, 98 Angiogenesis A108, A155, A171, A200, A201, A214, A228, A265, A305, A349, A350, A359, A410, A47, A48, A480, A483, A519, A5, 181, 257, 281, 327, 328, 351, 374, 435, 495, 583, 584, 602, 683, 76, 78, 802, 808, 873, 9 Antibody A7, A73, A81, A86, A91, A8, A99, A9, A104, A108, A108, A109, A109, A111, A115, A119, A126, A128, A130, A135, A138, A141, A144, A151, A153, A159, A160, A163, A164, A164, A171, A172, A175, A176, A2, A187, A193, A193, A195, A195, A198, A199, A20, A209, A212, A225, A229, A230, A23, A233, A233, A235, A239, A239, A240, A242, A244, A244, A245, A245, A246, A247, A247, A24, A250, A256, A260, A261, A266, A267, A268, A269, A273, A275, A286, A288, A292, A293, A296, A299, A300, A307, A3, A308, A31, A318, A324, A35, A334, A334, A336, A338, A339, A342, A343, A344, A344, A346, A346, A351, A351, A353, A356, A360, A361, A363, A368, A369, A370, A371, A372, A372, A373, A374, A376, A377, A378, A378, A379, A379, A380, A380, A388, A391, A399, A399, A41, A402, A410, A410, A411, A412, A414, A417, A418, A419, A419, A420, A422, A422, A423, A426, A427, A428, A428, A430, A431, A432, A434, A434, A435, A446, A454, A455, A459, A463, A465, A468, A468, A469, A474, A481, A483, A50, A486, A487, A491, A502, A502, A504, A507, A507, A508, A510, A511, A512, A512, A516, 12, 122, 133, 141, 151, 16, 167, 17, 174, 181, 182, 184, 185, 188, 195, 203, 210, 215, 218, 227, 231, 237, 243, 250, 253, 261, 262, 265, 267, 268, 281, 282, 287, 288, 3, 305, 313, 315, 317, 318, 323, 325, 34, 342, 348, 369, 377, 378, 38, 384, 385, 387, 393, 394, 395, 399, 401, 402, 403, 404, 405, 406, 407, 41, 412, 421, 428, 429, 438, 439, 442, 443, 450, 453, 469, 472, 476, 478, 480, 483, 486, 499, 5, 501, 51, 519, 531, 55, 550, 552, 557, 561, 562, 569, 571, 572, 573, 576, 578, 586, 587, 590, 594, 603, 605, 607, 612, 615, 616, 617, 618, 619, 621, 622, 626, 629, 630, 631, 632, 633, 634, 635, 647, 653, 665, 666, 67, 670, 683, 684, 686, 687, 690, 693, 696, 697, 698, 699, 702, 703, 704, 711, 713, 714, 715, 718, 721, 722, 725, 726, 727, 745, 757, 760, 766, 772, 776, 780, 781, 785, 793, 804, 808, 81, 812, 814, 820, 842, 843, 846, 851, 852, 853, 856, 858, 860, 861, 869 Antigen presenting cells A67, A79, A80, A92, A95, A96, A99, A99, A101, A102, A102, A108, A108, A111, A129, A139, A160, A166, A177, A178, A183, A191, A191, A213, A220, A221, A245, A254, A256, A262, A266, A275, A314, A322, A327, A328, A35, A335, A345, A359, A359, A360, A361, A364, A366, A368, A369, A371, A372, A403, A409, A411, A411, A418, A422, A433, A449, A48, A468, A471, A472, A4, A50, A495, A502, A506, A513, A514, A515, 108, 128, 130, 153, 157, 160, 166, 167, 168, 169, 170, 181, 182, 188, 217, 235, 263, 271, 290, 291, 299, 310, 311, 349, 360, 362, 404, 418, 421, 431, 438, 453, 510, 526, 538, 540, 55, 553, 575, 601, 602, 603, 605, 610, 611, 612, 615, 617, 618, 672, 682, 685, 686, 695, 703, 724, 748, 78, 780, 787, 790, 8, 81, 828, 842, 849, 862, 864, 868 Autoimmunity A8, A116, A15, A180, A181, A22, A380, A387, A394, A395, A402, A408, A502, A510, 16, 197, 27, 295, 296, 37, 637, 645, 657, 658, 671, 680, 843, 856