Suggestions for “safe” residue substitutions in site-directed mutagenesis

Bordo, Domenico; Argos, Patrick

doi:10.1016/0022-2836(91)90528-e

Cited by 366 publications

(254 citation statements)

References 69 publications

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“…The residues that were substituted for the well-conserved Gly and Lys residues (GK) of Walker A, and the Asp and Glu residues (DE) of Walker B, were selected using a matrix designed for creating 'safe substitutions' that minimized protein instability (Fig. 2c) (Bordo & Argos, 1991). Within the Walker A region, the small, non-polar Gly was substituted with the negatively charged, polar Asp (G210D).…”

Section: Generation Of Ntp-binding Motif Mutations In Tragmentioning

confidence: 99%

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

et al. 2005

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Bacterial conjugation is a horizontal gene transfer event mediated by the type IV secretion system (T4SS) encoded by bacterial plasmids. Within the T4SS, the coupling protein plays an essential role in linking the membrane-associated pore-forming proteins to the cytoplasmic, DNA-processing proteins. TraG is the coupling protein encoded by the incompatibility group HI plasmids. A hallmark feature of the IncHI plasmids is optimal conjugative transfer at 30 6C and an inability to transfer at 37 6C. Transcriptional analysis of the transfer region 1 (Tra1) of R27 has revealed that traG is transcribed in a temperature-dependent manner, with significantly reduced levels of expression at 37 6C as compared to expression at 30 6C. The R27 coupling protein contains nucleoside triphosphate (NTP)-binding domains, the Walker A and Walker B boxes, which are well conserved among this family of proteins. Site-specific mutagenesis within these motifs abrogated the conjugative transfer of R27 into recipient cells. Mutational analysis of the TraG periplasmic-spanning residues, in conjunction with bacterial two-hybrid and immunoprecipitation analysis, determined that this region is essential for a successful interaction with the T4SS protein TrhB. Further characterization of TraG by immunofluorescence studies revealed that the R27 coupling protein forms membrane-associated fluorescent foci independent of R27 conjugative proteins. These foci were found at discrete positions within the cell periphery. These results allow the definition of domains within TraG that are involved in conjugative transfer, and determination of the cellular location of the R27 coupling protein.

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Section: Generation Of Ntp-binding Motif Mutations In Tragmentioning

confidence: 99%

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

et al. 2005

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“…We also replaced the highly conserved His #(* residue individually with serine, resulting in WecA H#(*S . Serine was used for the single replacement because it is considered to be a conservative substitution for histidine (Bordo & Argos, 1991). Plasmids harbouring wecA FLAG (pAA26), as well as the mutated wecA HIHH/GGGG (pAA33) and wecA H#(*S (pAA51) genes were transformed into E. coli MV501.…”

Section: Conserved Residues In the Predicted Large Cytosolic Loop Of mentioning

confidence: 99%

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

Amer¹,

Valvano²

2001

Microbiology

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“…Although substitutions of aromatic residues with Leu are usually tolerated during evolution (Bordo & Argos, 1991), single replacements of this type have been found to destabilize folded proteins significantly (Kellis et al, 1988;Goldenberg et al, 1989;Coplen et al, 1990;Eriksson et al, 1993). The replacement decreases the size of the side chain and eliminates any interaction specific for aromatic rings.…”

Section: Design and Initial Characterization Of Aromatic + Leu Bpti Vmentioning

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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Suggestions for “safe” residue substitutions in site-directed mutagenesis

Cited by 366 publications

References 69 publications

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

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

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