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1.
Oncogene ; 28(5): 762-72, 2009 Feb 05.
Article in English | MEDLINE | ID: mdl-19015633

ABSTRACT

High-risk mucosal human papillomaviruses (HPV), mainly HPV16 and HPV18, are implicated in cervical carcinogenesis. HPV16 E6 oncoprotein binds and often targets for degradation numerous cell proteins, including the tumor suppressor p53 and several PDZ domain proteins. Here, we show that a single-point mutation, F47R, is sufficient to convert the HPV16 E6 oncoprotein into a suppressor of HPV-positive HeLa cervical cancer cells proliferation. The E6 F47R mutant is defective for polyubiquitination and subsequent degradation of p53. When expressed in HPV-positive cervical cancer cells, E6 F47R acts as a dominant negative mutant by counteracting the p53 degradation activity of endogenous E6 and restoring high p53 protein levels. Moreover, the prolonged expression of E6 F47R leads to suppression of HeLa cells proliferation through the induction of premature senescence. This phenotype is independent on the PDZ-binding activity of E6. F47R-senescent HeLa cells exhibit a sustained expression of p53, hMDM2 and p21(CIP) proteins and a reduced expression of endogenous HPV18 E6 protein. Finally, small interfering RNAs directed against p53 counteract the effect of E6 F47R expression, indicating that E6 F47R-induced cellular senescence is strongly dependent on p53 signaling pathway.


Subject(s)
Carcinoma/pathology , Cellular Senescence/genetics , Genes, Tumor Suppressor , Mutation/physiology , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/physiology , Papillomavirus Infections/pathology , Repressor Proteins/genetics , Repressor Proteins/physiology , Tumor Suppressor Protein p53/physiology , Uterine Cervical Neoplasms/pathology , Carcinoma/genetics , Cell Proliferation , Codon/genetics , Female , Genes, Dominant , Genes, Tumor Suppressor/physiology , HeLa Cells , Human papillomavirus 16/genetics , Humans , Papillomavirus Infections/genetics , Protein Processing, Post-Translational , Protein Stability , Signal Transduction/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/genetics
2.
Acta Crystallogr D Biol Crystallogr ; 62(Pt 10): 1150-61, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17001092

ABSTRACT

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Proteomics/methods , Algorithms , Data Interpretation, Statistical , Models, Molecular , Proteins/chemistry
3.
Protein Expr Purif ; 23(1): 22-32, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11570842

ABSTRACT

Many polypeptides overexpressed in bacteria are produced misfolded and accumulate as solid structures called inclusion bodies. Inclusion-body-prone proteins have often been reported to escape precipitation when fused to maltose-binding protein (MBP). Here, we have examined the case of HPV 16 oncoprotein E6. The unfused sequence of E6 is overexpressed as inclusion bodies in bacteria. By contrast, fusions of E6 to the C-terminus of MBP are produced soluble. We have analyzed preparations of soluble MBP-E6 fusions by using three independent approaches: dynamic light scattering, lateral turbidimetry, and sandwich ELISA. All three methods showed that MBP-E6 preparations contain highly aggregated material. The behavior of these soluble aggregates under denaturating conditions suggests that they are formed by agglomeration of misfolded E6 moieties. However, precipitation is prevented by the presence of the folded and highly soluble MBP moieties, which maintain the aggregates in solution. Therefore, the fact that a protein or protein domain is produced soluble when fused to the C-terminus of a carrier protein does not guarantee that the protein of interest is properly folded and active. We suggest that aggregation of fusion proteins should be systematically assayed, especially when these fusions are to be used for binding measurements or activity tests.


Subject(s)
Carrier Proteins/pharmacology , Inclusion Bodies/chemistry , Oncogene Proteins, Viral/chemistry , Recombinant Fusion Proteins/chemistry , Repressor Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Dimerization , Enzyme-Linked Immunosorbent Assay , Humans , Inclusion Bodies/drug effects , Inclusion Bodies/metabolism , Maltose-Binding Proteins , Nephelometry and Turbidimetry , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Protein Denaturation/drug effects , Protein Folding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Scattering, Radiation , Solubility/drug effects , Transformation, Genetic
4.
Protein Eng ; 14(4): 297-305, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11391022

ABSTRACT

Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E6 to the C-terminus of carrier proteins such as maltose-binding protein or glutathione-S-transferase are produced soluble. However, it has not yet been possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparations. We find that soluble MBP-E6 preparations contain two subpopulations. A major fraction is aggregated and contains exclusively misfolded E6 moieties ('soluble inclusion bodies'). A minor fraction is monodisperse and contains the properly folded E6 moieties. Using monodispersity as a screening criterion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to aggregated MBP-E6, these preparations yielded fully soluble E6 after proteolytic removal of MBP. Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measurements on pure E6 were performed. This work shows that solubility is not a sufficient criterion to check that the passenger protein in a fusion construct is properly folded and active. By contrast, monodispersity appears as a better quality criterion. The monodispersity-based strategy presented here constitutes a general method to prepare fusion proteins with optimized folding and biological activity.


Subject(s)
Oncogene Proteins, Viral/chemistry , Repressor Proteins , Viral Fusion Proteins/chemistry , Carrier Proteins/pharmacology , Dimerization , Glutathione Transferase/pharmacology , Humans , Inclusion Bodies, Viral/metabolism , Maltose-Binding Proteins , Papillomaviridae/chemistry , Protein Folding , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Solubility , Viral Fusion Proteins/isolation & purification
5.
J Mol Biol ; 305(4): 729-39, 2001 Jan 26.
Article in English | MEDLINE | ID: mdl-11162088

ABSTRACT

E6 is an oncoprotein implicated in cervical cancers produced by " high risk " human papillomaviruses. E6 binds specifically to several cellular proteins, including the tumour suppressor p53 and the ubiquitin ligase E6-AP. However, E6 is also a DNA-binding protein which recognizes a structural motive present in four-way junctions. Here, we demonstrate that the C-terminal zinc-binding domain of E6, expressed separately from the rest of the protein, fully retains the selective four-way junction recognition activity. The domain can bind to two identical and independent sites on a single junction, whereas full-length E6 can only bind to one site. The junction bound to either one or two domains adopts an extended square conformation. These results allow us to assign the structure-dependent DNA recognition activity of E6 to its C-terminal domain, which therefore represents a new class of zinc-stabilized DNA-binding module. Comparison with the binding characteristics of other junction-specific proteins enlightens the rules which govern protein-induced deformation of four-way DNA junctions.


Subject(s)
DNA/metabolism , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/metabolism , Papillomaviridae , Recombination, Genetic/genetics , Repressor Proteins , Zinc/metabolism , Amino Acid Sequence , Binding Sites , DNA/chemistry , DNA/genetics , DNA Probes/chemistry , DNA Probes/genetics , DNA Probes/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Oncogene Proteins, Viral/genetics , Papillomaviridae/chemistry , Papillomaviridae/genetics , Papillomaviridae/pathogenicity , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Substrate Specificity
6.
J Mol Biol ; 296(5): 1189-203, 2000 Mar 10.
Article in English | MEDLINE | ID: mdl-10698626

ABSTRACT

E6 is an oncoprotein implicated in cervical cancers, produced by "high-risk" human papillomaviruses. E6 is thought to promote tumorigenesis by stimulating cellular degradation of the tumour suppressor p53, but it might display other activities. Sequence similarity was recently detected between E6 and endonuclease VII, a protein of phage T4 that recognizes and cleaves four-way DNA junctions. Here, we purified recombinant E6 proteins and demonstrated that high-risk E6 s bind selectively to four-way junctions in a structure-dependent manner. Several residues in the C-terminal zinc-binding domain, the region of E6 similar to endonuclease VII, are necessary for the junction-binding activity. E6 binds to the junction as a monomer. Comparative electrophoresis shows that E6-bound junctions migrate in an extended square conformation. Magnesium inhibits the electrophoretic migration of the complexes but does not seem to influence their formation at equilibrium. This work is the first demonstration of specific binding of purified active E6 to a well-characterized DNA ligand, and suggests new modes of action of E6 in oncogenesis.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Nucleic Acid Conformation , Oncogene Proteins, Viral/metabolism , Amino Acid Sequence , Binding Sites/drug effects , DNA/chemical synthesis , DNA/genetics , DNA Probes/chemical synthesis , DNA Probes/chemistry , DNA Probes/genetics , DNA Probes/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Magnesium/pharmacology , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Conformation/drug effects , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/isolation & purification , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomaviridae/pathogenicity , Protein Structure, Quaternary , Protein Structure, Tertiary/drug effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Static Electricity , Substrate Specificity , Thermodynamics , Tumor Suppressor Protein p53/metabolism , Zinc/metabolism
7.
J Mol Recognit ; 12(2): 141-52, 1999.
Article in English | MEDLINE | ID: mdl-10398405

ABSTRACT

The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein.


Subject(s)
Antibodies, Monoclonal/pharmacology , Immunoglobulin Fragments/pharmacology , Oncogene Proteins, Viral/antagonists & inhibitors , Papillomaviridae/physiology , Repressor Proteins , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Carcinoma, Squamous Cell/pathology , Epitopes/chemistry , Epitopes/immunology , Female , Humans , Immunoglobulin Fragments/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/immunology , Oncogene Proteins, Viral/pharmacology , Recombinant Fusion Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Uterine Cervical Neoplasms/pathology
8.
EMBO J ; 14(20): 4922-31, 1995 Oct 16.
Article in English | MEDLINE | ID: mdl-7588621

ABSTRACT

Calcium is a universally employed cytosolic messenger in eukaryotic cells. Most of the proteins that bind signalling calcium are members of the calmodulin superfamily and share two or more helix-loop-helix motifs known as EF-hands. A model, based on structure comparison of different domains and supported by preliminary NMR data, has suggested that EF-hands involved in signal transduction undergo a major conformational change upon calcium binding from a 'closed' to an 'open' state allowing protein-protein interaction. We have determined the solution structures of the EF-hand pair from alpha-spectrin in the absence and in the presence of calcium. The structures are in the closed and open conformation respectively, providing a definite experimental proof for the closed-to-open model. Our results allow formulation of the rules which govern the movement induced by calcium. These rules may be generalized to other EF-hands since the key residues involved are conserved within the calmodulin family.


Subject(s)
Calcium/pharmacology , Spectrin/chemistry , Amino Acid Sequence , Calcium/metabolism , Computer Simulation , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Conformation/drug effects , Sequence Homology, Amino Acid , Spectrin/drug effects , Spectrin/metabolism , Troponin/chemistry , Troponin C
9.
Eur J Biochem ; 227(1-2): 35-42, 1995 Jan 15.
Article in English | MEDLINE | ID: mdl-7851407

ABSTRACT

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF-hand calcium-binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C-terminus of alpha-spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0.1-1.0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N-terminal and C-terminal halves which fold independently. Only the N-terminal subdomain binds calcium. These data suggest that the C-terminus of alpha-spectrin has a domain with a calmodulin fold and two calcium-binding sites. Sequence alignments suggest that the related domains in alpha-actinin, and possibly in dystrophin, may share the same calmodulin-like structure. However, only non-muscle alpha-actinins appear to have one or two EF-hand(s) with the calcium-binding consensus sequence, and a strict consensus is not found in the muscle alpha-actinins or dystrophins.


Subject(s)
Calmodulin/chemistry , Spectrin/chemistry , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calcium/metabolism , Cloning, Molecular , DNA Primers , Escherichia coli/genetics , Humans , Molecular Sequence Data , Protein Folding , Sequence Homology, Amino Acid , Spectrin/genetics , Spectrin/isolation & purification , Spectrin/metabolism
10.
FEBS Lett ; 358(2): 193-8, 1995 Jan 23.
Article in English | MEDLINE | ID: mdl-7828735

ABSTRACT

The KH module has recently been identified in a number of RNA associated proteins including vigilin and FMR1, a protein implicated in the fragile X syndrome. In this work, NMR spectroscopy was used to determine the secondary structure in solution of a KH domain (repeat 5 from vigilin). Almost complete assignments were obtained for the 1H and 15N resonances using uniform 15N-labeling of the protein combined with homo-nuclear 2D 1HNMR and 3D 15N correlated 1H NMR. On the basis of NOE patterns, secondary chemical shifts and amide solvent exposure, the secondary structure consists of an antiparallel three stranded beta sheet connected by two helical regions. This domain may also be stabilized by an appended C-terminal helix which is common to many but not all members of the KH family.


Subject(s)
Carrier Proteins , Proteins/chemistry , RNA-Binding Proteins , Amino Acid Sequence , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Structure, Secondary
11.
Protein Eng ; 7(5): 689-96, 1994 May.
Article in English | MEDLINE | ID: mdl-7915419

ABSTRACT

All the functions of annexins in vitro as well as in vivo are mediated and probably regulated by calcium. We have used recombinant annexin I, synthesized by Escherichia coli, and we have performed site-directed mutagenesis. We have mutated the endonexin fold of domain 2 that binds calcium. Mutations were performed in this domain of the molecule because it perfectly matches the calcium binding consensus sequence. The two glycines of this fold were mutated into glutamic acid. The helix content and the stability of the mutants are identical to those of the wild-type, suggesting that the mutations did not drastically affect the structure of the protein. The two mutants showed modified calcium binding affinities. However, the calcium binding affinity of the G131E mutant was far more altered than that of the G129E mutant. Furthermore, other biochemical properties of these mutants were modified to different extents. The binding to phospholipid was not seriously affected, whereas the self-association was lost by the G131E mutant. In the same way, liposome aggregation is conserved, but modified, while the calcium affinity measured by equilibrium dialysis is dramatically altered.


Subject(s)
Annexin A1/chemistry , Annexin A1/metabolism , Calcium/metabolism , Mutagenesis, Site-Directed , Annexin A1/genetics , Binding Sites , Calcium/pharmacology , Dialysis , Drug Stability , Escherichia coli/genetics , Glutamates , Glutamic Acid , Glycine , Liposomes/metabolism , Models, Molecular , Molecular Structure , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structure-Activity Relationship , Thermodynamics
12.
Biochim Biophys Acta ; 1205(2): 215-22, 1994 Apr 13.
Article in English | MEDLINE | ID: mdl-8155700

ABSTRACT

We have mutated the lysine 128 of domain II of annexin I, which flanks a putative calcium-binding loop, into a glutamic acid residue. The properties of the mutated recombinant protein were compared to those of the wild-type recombinant protein. A change in the isotherm of calcium binding in the presence of lipids was observed. A slight decrease in the affinity for lipids was evident. When tested for the vesicle aggregation property, the mutation induced a change in lipid specificity; unlike the wild-type protein, the mutant protein aggregates vesicles containing phosphatidylserine plus phosphatidylethanolamine better than vesicles containing only phosphatidylserine. These experiments are in agreement with a model which suggests that a lipid molecule is inserted into the calcium-binding loop of annexin I and that the conserved lysine residue is involved in the specificity of annexins for anionic phospholipids.


Subject(s)
Annexin A1/metabolism , Calcium/metabolism , Phospholipids/metabolism , Amino Acid Sequence , Annexin A1/drug effects , Annexin A1/genetics , DNA Mutational Analysis , Liposomes/metabolism , Lysine/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylethanolamines/pharmacology , Phosphatidylserines/pharmacology , Protein Binding , Recombinant Proteins/metabolism , Structure-Activity Relationship
13.
Eur J Immunol ; 23(1): 179-85, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8419169

ABSTRACT

Jacalin is a plant lectin known to specifically induce the proliferation of CD4+ T lymphocytes in human. We demonstrate here that jacalin completely blocks human immunodeficiency virus type 1 (HIV-1) in vitro infection of lymphoid cells. Jacalin does not bind the viral envelope glycoprotein gp120. Besides other T cell surface molecules, it interacts with CD4, the high-affinity receptor to HIV. Binding of jacalin to CD4 does not prevent gp120-CD4 interaction and does not inhibit virus binding and syncytia formation. The anti-HIV effect of the native lectin can be reproduced by its separated alpha-subunits. More importantly, we have defined in the alpha-chain of jacalin a 14-amino acid sequence which shows high similarities with a peptide of the second conserved domain of gp120. A synthetic peptide corresponding to this similar stretch also exerts a potent anti-HIV effect. This peptide is not mitogenic for peripheral blood mononuclear cells and does not inhibit anti-CD3-induced lymphocyte proliferation. These results make jacalin alpha chain-derived peptide a potentially valuable therapeutic agent for acquired immunodeficiency syndrome.


Subject(s)
HIV Envelope Protein gp120/chemistry , HIV-1/drug effects , Lectins/pharmacology , Peptide Fragments/pharmacology , Plant Lectins , Amino Acid Sequence , Base Sequence , CD4 Antigens/metabolism , Cell Line , Cells, Cultured , HIV Envelope Protein gp120/metabolism , Humans , Molecular Sequence Data , Peptide Fragments/chemistry
14.
FEBS Lett ; 293(1-2): 34-6, 1991 Nov 18.
Article in English | MEDLINE | ID: mdl-1835701

ABSTRACT

Annexin 1 has been proposed to inhibit phospholipase A2 by direct interaction through a specific amino acid sequence spanning residues 246-254. The possible role of this region was investigated by protein engineering. Three point mutations and a deletion have been performed. The four mutant proteins have been expressed in E. coli, purified and tested for calcium and lipid binding, and for phospholipase inhibition. All mutant proteins conserved the properties of the wild-type recombinant protein. This result clearly demonstrates that this part of the molecule is not involve in the inhibition of phospholipase A2.


Subject(s)
Calcium-Binding Proteins/genetics , Phospholipases A/antagonists & inhibitors , Amino Acid Sequence , Annexins , Base Sequence , Calcium-Binding Proteins/pharmacology , Cell Line , DNA Mutational Analysis , Humans , Molecular Sequence Data , Phospholipases A2 , Protein Engineering
15.
Biochimie ; 72(1): 19-24, 1990 Jan.
Article in English | MEDLINE | ID: mdl-2111175

ABSTRACT

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.


Subject(s)
Calmodulin/metabolism , Carrier Proteins/metabolism , Microfilament Proteins/metabolism , Spectrin/metabolism , Amino Acid Sequence , Base Sequence , Carrier Proteins/genetics , Microfilament Proteins/genetics , Molecular Sequence Data , Mutation , Protein Conformation , Spectrin/genetics
16.
J Mol Biol ; 205(2): 455-8, 1989 Jan 20.
Article in English | MEDLINE | ID: mdl-2926814

ABSTRACT

We have screened a lambda gt11 library, constructed with mouse macrophage cDNA, in order to isolate clones that code for calmodulin binding proteins. We have developed a new approach for this purpose using radioactive calmodulin (produced by genetic engineering) to detect fusion proteins that interact with this protein with high affinity. A cDNA clone that codes for mouse macrophage fodrin was isolated, sequenced and identified. By deleting part of the sequence the calmodulin binding domain was located on the fodrin sequence. The site is situated on repeat 11 of fodrin and probably on the extra arm of this repeat. The method we developed is widely applicable to site-directed mutagenesis of interacting proteins.


Subject(s)
Calmodulin/metabolism , Carrier Proteins/metabolism , Microfilament Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chromosome Deletion , Cloning, Molecular , Mice , Molecular Sequence Data , Mutation
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