TAT-aFGF融合蛋白在大肠杆菌中的表达及其生物活性测定

构建表达Tat与人酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）融合蛋白的重组质粒pET3c–Tat-aFGF14-154和pET3c-Tat-aFGF27-154,并转化大肠杆菌BL21（DE3）中。37℃,l mmol/L的IPTG诱导4 hrs后,获得分子量约为18 kDa的融合蛋白,表达量分别占菌体总蛋白的42%和24%。利用阳离子交换（CM-Sepharose FF）、肝素亲和层析（Heparin-Sepharose CL-6B）以及分子筛（Sephadex G-25）联用的方法可获得纯度大于95%的目的蛋白,得率分别为93和78 mg/L。Tat-aFGF14-154及其对照蛋白aFGF14-154对Balb/c 3T3细胞有明显促细胞增殖的作用,最佳作用浓度分别为1280 ng/ml和160 ng/ml。而Tat-aFGF27-154 及其对照蛋白aFGF27-154则几乎没有促细胞增殖活性。免疫荧光分析结果表明,Tat-aFGF14-154及Tat-aFGF27-154均能穿过PC12、Balbc/3T3、HaCaT和海马神经元细胞的细胞膜,并主要定位于细胞浆中。此外,四种重组蛋白均能降低Aβ25-35对大鼠海马神经元细胞的毒性,在剂量为1000 ng/ml时,Tat-aFGF14-154、Tat-aFGF27-154、aFGF14-154及aFGF27-154细胞存活率分别为90.66%、81.87%、85.71%和78.02%,而Aβ25-35模型组的存活率仅为56.87%。     

The plasma cell membrane glycoprotein, PC-1, is a threonine-specific protein kinase stimulated by acidic fibroblast growth factor.

A 32P-labeled protein that co-purified with acidic fibroblast growth factor (aFGF) receptor from bovine liver proved to be a distinct membrane protein, which itself has kinase activity that is stimulated by aFGF. The protein was designated MAFP for major aFGF-stimulated phosphoprotein. MAFP was purified from bovine liver using immunoaffinity chromatography with monoclonal antibody to MAFP following Triton X-100 extraction of plasma membranes and wheat germ lectin-Sepharose 4B column chromatography. The purified MAFP showed molecular masses of 130 kDa and 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. Purified MAFP elicited aFGF-stimulated Thr-specific autophosphorylation activity and phosphorylation activity toward protein substrates (myelin basic protein and histone). Amino acid sequence analyses of 16 peptide fragments of MAFP, produced by endoproteinase Lys-C digestion followed by reduction and S-pyridylethylation, showed approximately 80-100% homology with the cDNA-deduced amino acid sequences of human and mouse plasma cell membrane glycoprotein, PC-1 (Buckley, M. F., Loveland, K. A., McKinstry, W. J., Garson, O. M., and Goding, J. W. (1990) J. Biol. Chem. 265, 17506-17511), suggesting that MAFP is the bovine version of PC-1. The amino acid sequences of bovine MAFP, human and mouse PC-1 reveal a putative ATP binding site in their extracellular domains. These results suggest that MAFP(PC-1) is an ectoprotein kinase. In addition to the kinase activity, MAFP(PC-1) was also found to possess alkaline nucleotide phosphodiesterase activity. It is now clear that several of the unique properties previously attributed to the aFGF receptor kinase are actually properties of this novel Thr-specific ectoprotein kinase, which co-purifies with the aFGF receptor and is responsive to stimulation by aFGF.     

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions.

A highly efficient expression for human acidic fibroblast growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.     

High-level expression and purification of a nonmitogenic form of human acidic fibroblast growth factor in Escherichia coli

To decrease the potential side effects of acidic fibroblast growth factor (aFGF) caused by its broad-spectrum mitogenic activity, a nonmitogenic form of aFGF (nhaFGF), which retained the cardio- and neuroprotective characters of the wild-type aFGF, was overexpressed in Escherichia coli. The expression level of nhaFGF was up to 25% of the total cellular protein. The expressed nhaFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. The mitogenic activity of the purified nhaFGF was decreased dramatically comparable to that of the wild-type aFGF (haFGF) detected by methylthiazoletetrazolium method. The purified recombinant nhaFGF was sufficiently prepared and sufficient for the following pharmacological study.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

Selection of peptide ligands that bind to acid fibroblast growth factor

Acid fibroblast growth factor (aFGF) binds to its cell-surface receptors in a heparin-dependent manner. In an attempt to define the aFGF recognition site on fibroblast growth factor receptor 1 (FGFR1), we developed a screening strategy for identifying FGF ligands that bind to the receptor-binding region of FGF. To retain the natural conformation of aFGF during screening, we used biotinylated heparin to immobilize aFGF on a streptavidin-coated dish. A 15-mer phage display peptide library was then screened in the dish and a group of related peptide sequences was identified. These peptide sequences contain two conserved motifs, SSG and VPS, corresponding to two protein sequences of the immunoglobulin-like (Ig-like) domain II of FGFR1 at amino acids 180-182 and 221-223 (CPSSG-VPSDKGNYTC). Further experiments demonstrate that the phage displaying these sequences can specifically bind to aFGF and that the synthesized peptide corresponding in sequence can inhibit mitogenic activity of aFGF. These sequences may thus constitute part of the aFGF-binding region on FGFR1, and the synthesized peptide has the potential to become a therapeutic agent as an aFGF antagonist.     

Effects of mutations of a phosphorylation site in an exposed loop in acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) contains a phosphorylation site recognized by protein kinase C. A non-mitogenic mutant growth factor is devoid of this phosphorylation site. We have changed amino acids in and close to the phosphorylation site and studied the consequences of this for binding of the growth factor to high affinity receptors as well as to heparin. We have also studied the ability of the mutants to stimulate DNA synthesis and cell proliferation as well as phosphorylation of mitogen-activated protein kinase and the ability of the growth factor mutants to be transported to the nucleus. The results indicate that while the mutations strongly affect the ability of the growth factor to bind to heparin, they do not affect much the binding to the specific FGF receptors, activation of mitogen-activated protein kinase or transport of the growth factor to the nucleus. The mutations affect to various extents the ability of the growth factor to stimulate DNA synthesis and to induce cell multiplication. We find that phosphorylation of aFGF is not required for mitogenic activity. The data suggest that altered interaction of the growth factor with a cellular component different from the receptor, possibly a component in the nucleus, is the reason for the different mitogenicity of the different growth factor mutants.     

Complete primary structure of prostatropin, a prostate epithelial cell growth factor

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.     

Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is a broad spectrum mitogen that is stabilized by complexation with heparin and heparan proteoglycans. The monomeric human protein contains 3 reduced cysteine residues of unknown function, the first 2 of which are conserved among all seven known fibroblast growth factors. The influence of these free sulfhydryl groups on the level, stability, and heparin dependence of the mitogenic activity at physiological temperature and pH is characterized using a complete set of site-directed mutants in which either any 1, 2, or all 3 of the cysteine residues are converted to serines. Mutants of aFGF in which either any 2 or all 3 cysteine residues are substituted by serines are more active, have longer activity half-lives, and are less heparin dependent than wild-type aFGF. In contrast, wild-type aFGF and the three mutants that each retain 2 cysteine residues inactivate more rapidly in the absence of heparin by a nonproteolytic mechanism but are markedly stabilized by heparin. This cysteine-mediated destabilization of aFGF not only diminishes its activity in the absence of heparin in tissue culture but also could functionally restrict its activity in vivo to the vicinity of mast cell-derived heparins and heparan proteoglycans associated with cell surfaces and basement membranes.     

The mitogenic function of hepatitis B virus X protein resides within amino acids 51 to 140 and is modulated by N- and C-terminal regulatory regions

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. pX variants encoded by HBV genomes found integrated in genomic DNA from liver tumors of patients with hepatocellular carcinoma (HCC) generally lack amino acids 134 to 154. Since deregulation of mitogenic pathways is linked to oncogenic transformation, herein we define the pX region required for mitogenic pathway activation. A series of pX deletions was used to construct tetracycline-regulated pX-expressing cell lines. The activation of the mitogenic pathways by these pX deletions expressed in the constructed cell lines was measured by transient transreporter assays, effects on endogenous cyclin A expression, and apoptosis. Conditional expression of pX51-140 in AML12 clone 4 cell line activates the mitogenic pathways, induces endogenous cyclin A expression, and sensitizes cells to apoptosis, similar to wild-type (WT) pX. By contrast, pX1-115 is inactive, supporting the idea that amino acids 116 to 140 are required for mitogenic pathway activation. Moreover, this pX deletion analysis demonstrates that WT pX function is modulated by two regions spanning amino acids 1 to 78 and 141 to 154. The N-terminal X1-78, expressed via a retroviral vector in WT pX-expressing 4pX-1 cells, coimmunoprecipitates with WT pX, indicating this pX region participates in protein-protein interactions leading to pX oligomerization. Interestingly, pX1-78 interferes with WT pX in mediating mitogenic pathway activation, endogenous gene expression, and apoptosis. The C-terminal pX region spanning amino acids 141 to 154 decreases pX stability, determined by pulse-chase studies of WT pX and pX1-140, suggesting that increased stability of naturally occurring pX variants lacking amino acids 134 to 154 may play a role in HCC development.     

The use of mutants in the study of structure-function relationships in cloacin DF13

A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03· immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated. Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent. Comparison of some physicochemical characteristics of the cloacin clp03 · immunity protein complex and wild-type cloacin complex showed no significant differences.From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity.Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues. Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein. Since the immunity protein probably not only provides cloacin-producing cells with “immunity” but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82.The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al. (de Graaf, F.K., Stukart, M.J., Boogerd, F.C. and Metselaar, K. (1978) Biochemistry, in press) are discussed.     

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.     

The use of mutants in the study of structure-function relationships in cloacin DF13.

A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03 . immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated. Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent. Comparison of some physicochemical characteristics of the cloacin clp03 . immunity protein complex and wild-type cloacin complex showed no significant differences. From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity. Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues. Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein. Since the immunity protein probably not only provides cloacin-producing cells with "immunity" but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82. The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al. (de Graaf, F.K., Stukart, M.J., Boogerd, F.C. and Metselaar, K. (1978) Biochemistry, in press) are discussed.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

Ligand Binding by Fibroblast Growth Factor Receptors Investigated Using Chimeric Receptor Molecules

Abstract

In order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.     

Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells

Nerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK-21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.     

Phosphatidylinositol linkage of a truncated form of the platelet-derived growth factor receptor

The platelet-derived growth factor (PDGF) receptor is usually anchored to the plasma membrane through a membrane-spanning hydrophobic amino acid sequence that splits the molecule into two approximately equal pieces, an amino-terminal external domain that contains the binding site for PDGF and a carboxyl-terminal cytoplasmic domain that includes the tyrosine kinase coding sequences. Here we report the expression of a truncated PDGF receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. Unexpectedly, this form of the receptor that lacks a hydrophobic membrane-anchoring sequence was bound to the membrane and was not secreted into the culture media. Conventional methods to dissociate noncovalent protein-protein interactions failed to release the protein from the membrane. When the transmembrane and cytoplasmic sequences were artificially deleted from the PDGF receptor, the truncated extracellular domain was anchored to the membrane through phospholipids and could be released by phospholipase C treatment. This truncated form of the receptor bound PDGF with an affinity 5-20-fold lower than the full-length receptor.     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120 A distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.     

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: A bona fide tracer

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF–Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group’s side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF–Cys2–heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF–Cys2–heparin complex might represent a biologically relevant complex in physiological media.