Rapid and efficient site-specific mutagenesis without phenotypic selection.

Kunkel, Thomas A.

doi:10.1073/pnas.82.2.488

Cited by 6,748 publications

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“…Single Cys mutants of the human cPLA 2 α C2 domain (residues 1-138) were generated using the single-stranded mutagenesis technique of Kunkel et al as previously described (25,26). Cys mutants were expressed as inclusion bodies in Escherichia coli, then were isolated, denatured, and refolded (26).…”

Section: Methods Protein Mutagenesis Expression and Purificationmentioning

confidence: 99%

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

et al. 2004

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During Ca 2+ activation, the Ca 2+ -binding sites of C2 domains typically bind multiple Ca 2+ ions in close proximity. These binding events exhibit positive cooperativity, despite the strong charge repulsion between the adjacent divalent cations. Using both experimental and computational approaches, the present study probes the detailed mechanisms of Ca 2+ activation and positive cooperativity for the C2 domain of cytosolic phospholipase A 2 , which binds two Ca 2+ ions in sites I and II, separated by only 4.1 Å. First, each of the five coordinating side chains in the Ca 2+ -binding cleft is individually mutated and the effect on Ca 2+ -binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca 2+ affinity, while the two coordinating side chains that provide bridging coordination to both Ca 2+ ions, Asp 43 and Asp 40, are observed to make the largest contributions to positive cooperativity. Electrostatic calculations reveal that Asp 43 possesses the highest pseudo-pK a of the coordinating acidic residues, as well as the highest general cation affinity, due to its relatively buried location within 3.5 Å of seven protein oxygens with full or partial negative charges. These calculations therefore explain the greater importance of Asp 43 in defining the Ca 2+ affinity. Overall, the experimental and computational results support an activation model in which the first Ca 2+ ion binds usually to site I, thereby preordering both bridging side chains Asp 40 and 43, and partially or fully deprotonating the three coordinating Asp residues. This initial binding event prepares the conformation and protonation state of the remaining site for Ca 2+ binding, enabling the second Ca 2+ ion to bind with higher affinity than the first as required for positive cooperativity.The C2 domain is a ubiquitous membrane-targeting protein module found in a diverse array of signal transducing proteins that regulate key cellular processes at membrane surfaces (reviewed in refs 1-10). These processes include the generation of lipid-derived second messengers, vesicular targetting and fusion, GTPase regulation, protein phosphorylation, pore formation by cytolytic T cells, and ubiquitin-mediated protein degradation. The majority of C2 domains are activated by cytoplasmic Ca 2+ signals and dock to specific membrane-associated targets such as phospholipids or, less commonly, to membrane-bound proteins.The structures of representative Ca 2+ -regulated C2 domains, including the C2A domain of synaptotagmin I (Syt-IA), 1 the C2 domain of cytosolic phospholipase A 2 (α-isoform, cPLA 2 α), and the C2 domain of protein kinase C (PKC), have been determined by X-ray † Support provided by NIH Grants GM R01-063235 (to J.J.F.) and GM R01-054651 (to H.L.S./E.J.), and by a DOE/GTL Grant (to E.J.).

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Section: Methods Protein Mutagenesis Expression and Purificationmentioning

confidence: 99%

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

et al. 2004

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“…Site-directed mutagenesis was performed according to Kunkel (1985). The oligonucleotides used in this study are listed in Table 3.…”

Section: Dna Manipulationsmentioning

confidence: 99%

Proline 21, a residue within the α‐helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

Witte

Schrot

Schön

et al. 1997

Molecular Microbiology

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Summary⌽X174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis-trans isomerization of proline residues within ␣-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded ␣-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli. Oligomerization of protein P21G-StrpA was not disturbed.

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“…The proteins were produced as fusions to the leader peptide of the OmpA protein, an E. coli outer membrane protein, and exported to the periplasmic space, where the leader peptide was removed and the polypeptides folded to yield native protein. Mutations in the BPTI coding sequence were constructed using the method of Kunkel (1985).…”

Section: Preparation Of Bpti Variantsmentioning

confidence: 99%

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

Zhang

Goldenberg

1997

Protein Science

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The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.Keywords: aromatic residues; bovine pancreatic trypsin inhibitor; conformational funnels; disulfide bonds; hydrophobic residues; protein foldingThe identification and characterization of partially folded intermediate states have historically been major goals in the study of protein folding mechanisms. Early experiments demonstrating the transient accumulation of such species provided important evidence supporting the view that proteins fold via non-random pathReprint requests to: David P. Goldenberg, Department of Biology, University of Utah, Salt Lake City, UT 84112; e-mail: goldenberg@ bioscience.utah.edu.'Current address: Department of Pathology, Harvard University Medical School, Boston, MA 021 15.Abbreviations: BPI?, bovine pancreatic trypsin inhibitor. Amino acid replacements are indicated by the wild-type residue type (using the oneletter code for the 20 standard amino acids), followed by the residue number and the mutant residue type. The disulfides of native BF'TI and folding intermediates are indicated by the residue numbers of the disulfidebonded cysteine residues; N;;, native-like two-disulfide intermediate containing the 30-51 and 5-55 disulfides. Other intermediates are indicated by square brackets enclosing the disulfide bonds they contain; GSSG, oxidized glutathione; DTT:;, the dithiol form of dithiothreitol; GuHCl, guanidinium chloride; IAEDANS, N-iodoacetyl-N"(5-sulfo-l-naphthyl)ethylenediami...

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Rapid and efficient site-specific mutagenesis without phenotypic selection.

Cited by 6,748 publications

References 20 publications

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

Proline 21, a residue within the α‐helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

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Rapid and efficient site-specific mutagenesis without phenotypic selection.

Cited by 6,748 publications

References 20 publications

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

Proline 21, a residue within the α‐helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

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Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity