Fragment complementation of calbindin D28k

被引:23
作者
Berggård, T
Thulin, E
Åkerfeldt, KS
Linse, S
机构
[1] Univ Lund, Ctr Chem, S-22100 Lund, Sweden
[2] Haverford Coll, Dept Chem, Haverford, PA 19041 USA
关键词
Ca2+-binding protein; calbindin D-28k; cold denaturation; EF-hand; protein reconstitution; spectroscopy; surface plasmon resonance;
D O I
10.1110/ps.9.11.2094
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Calbindin D-28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D-28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M-1, with association and dissociation rate constants of 2.7 x 10(4) M-1 s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha -helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D-28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha -helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D-28k.
引用
收藏
页码:2094 / 2108
页数:15
相关论文
共 43 条
  • [1] Ca2+-binding stoichiometry of calbindin D-28k as assessed by spectroscopic analyses of synthetic peptide fragments
    Akerfeldt, KS
    Coyne, AN
    Wilk, RR
    Thulin, E
    Linse, S
    [J]. BIOCHEMISTRY, 1996, 35 (12) : 3662 - 3669
  • [2] PROTEIN STABILITY AND ELECTROSTATIC INTERACTIONS BETWEEN SOLVENT EXPOSED CHARGED SIDE-CHAINS
    AKKE, M
    FORSEN, S
    [J]. PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1990, 8 (01): : 23 - 29
  • [3] ANDRIA G, 1971, J BIOL CHEM, V246, P7421
  • [4] Ca2+- and H+-dependent conformational changes of calbindin D28k
    Berggård, T
    Silow, M
    Thulin, E
    Linse, S
    [J]. BIOCHEMISTRY, 2000, 39 (23) : 6864 - 6873
  • [5] Structure of a calpain Ca2+-binding domain reveals a novel EF-hand and Ca2+-induced conformational changes
    Blanchard, H
    Grochulski, P
    Li, Y
    Arthur, JSC
    Davies, PL
    Elce, JS
    Cygler, M
    [J]. NATURE STRUCTURAL BIOLOGY, 1997, 4 (07) : 532 - 538
  • [6] COMPARATIVE STUDIES ON THERMOSTABILITY OF CALMODULIN, SKELETAL-MUSCLE TROPONIN-C AND THEIR TRYPTIC FRAGMENTS
    BRZESKA, H
    VENYAMINOV, SV
    GRABAREK, Z
    DRABIKOWSKI, W
    [J]. FEBS LETTERS, 1983, 153 (01) : 169 - 173
  • [7] CALMODULIN STRUCTURE REFINED AT 1.7 ANGSTROM RESOLUTION
    CHATTOPADHYAYA, R
    MEADOR, WE
    MEANS, AR
    QUIOCHO, FA
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1992, 228 (04) : 1177 - 1192
  • [8] CATION BINDING AND CONFORMATION OF TRYPTIC FRAGMENTS OF NEREIS SARCOPLASMIC CALCIUM-BINDING PROTEIN - CALCIUM-INDUCED HOMODIMERIZATION AND HETERODIMERIZATION
    DURUSSEL, I
    LUANRILLIET, Y
    PETROVA, T
    TAKAGI, T
    COX, JA
    [J]. BIOCHEMISTRY, 1993, 32 (09) : 2394 - 2400
  • [9] DISSECTION OF CALBINDIN-D(9K) INTO 2 CA2+-BINDING SUBDOMAINS BY A COMBINATION OF MUTAGENESIS AND CHEMICAL CLEAVAGE
    FINN, BE
    KORDEL, J
    THULIN, E
    SELLERS, P
    FORSEN, S
    [J]. FEBS LETTERS, 1992, 298 (2-3) : 211 - 214
  • [10] 3-DIMENSIONAL STRUCTURE OF RECOVERIN, A CALCIUM SENSOR IN VISION
    FLAHERTY, KM
    ZOZULYA, S
    STRYER, L
    MCKAY, DB
    [J]. CELL, 1993, 75 (04) : 709 - 716