Mutagenesis-based definitions and probes of residue burial in proteins

K. Bajaj, P. Chakrabarti, R. Varadarajan
2005 Proceedings of the National Academy of Sciences of the United States of America  
Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB
more » ... ranscriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, maltose-binding protein and thioredoxin, also were shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose-dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. With continuous reductions in oligonucleotide costs and increasingly efficient site-directed mutagenesis procedures, comprehensive scanning mutagenesis experiments for small pro-teins͞domains are quite feasible. accessibility ͉ aspartate ͉ scanning mutagenesis ͉ phenotype I t is well known that buried residues in a protein are important determinants of protein stability while surface residues are involved in protein function (1). Residue burial in a protein structure is typically quantitated in terms of accessible surface area and percent side-chain accessibility of a residue in a protein (ACC) (2). ACC is the accessible surface area of the residue relative to that found for the same residue in a Gly-X-Gly tripeptide of extended conformation, and burial is often equated to 100-ACC (3). There is no universally accepted definition of what ACC cutoff should be used to distinguish buried from surface residues, although a variety of values ranging from 5% to 25% have been used (4, 5). In the absence of a 3D structure, it is not always straightforward to determine the extent of residue burial experimentally. For residues with chemically reactive functional groups (such as Lys or Cys), it is possible to probe burial by examining the reactivity toward residue-specific labeling reagents. Solvent accessibilities of residues also can be characterized by using site-directed fluorescence labeling monitored by fluorescent anisotropy and lifetime measurements (6). However, labeling rates are often influenced by local geometries, the nature of surrounding residues, and other features besides side-chain burial. Hydrogen exchange is another powerful tool for studying protein structure and dynamics. Unfortunately, hydrogenexchange rates are poorly correlated with residue burial (7). Recently, a combination of synchrotron radiolysis and mass spectrometry (MS) has been used to provide qualitative information on residue burial and residues involved in protein:protein or protein:DNA interaction (8). In the present work, we examine the feasibility of using scanning mutagenesis to distinguish between buried and exposed positions and also to arrive at an experimental definition of the appropriate ACC cutoff to distinguish between buried and exposed residues. The experimental system, Controller of Cell Division or Death B protein (CcdB), is a 101-residue, homodimeric protein encoded by F plasmid. The protein is an inhibitor of DNA gyrase and is a potent cytotoxin in Escherichia coli (9). Crystallographic structures of CcdB in the free and gyrase bound forms (10, 11) are also available. Transformation of normal E. coli cells with plasmid expressing the wild-type (WT) ccdb gene results in cell death. If the protein is inactivated through mutation, cells transformed with such mutant genes will survive. Each residue of CcdB was replaced with Ala, Asp, Glu, Lys, and Arg. All mutants were expressed under the control of the P BAD promoter, which allows for dose-dependent protein expression by varying the amount of inducer added (12). CcdB phenotype was assayed as a function of expression level and residue burial by monitoring the presence or absence of cell growth. Methods Plasmids and Host Strains. The ccdb gene was cloned under the control of the arabinose P BAD promoter in the vector pBAD24 to yield the construct pBAD24CcdB (13). Three E. coli host strains were used, TOP10, XL1-Blue, and CSH501. TOP10 is sensitive to the action of CcdB and used for screening the phenotype. XL1-Blue is able to tolerate low levels of CcdB protein expression because of the presence of the antidote CcdA, which is encoded by the resident F plasmid and was used for plasmid propagation. CSH501 is completely resistant to the action of CcdB because the strain harbors the GyrA462 mutation in its chromosomal DNA and prevents gyrase from binding to CcdB. CSH501 was kindly provided by M. Couturier (Universite Libre de Bruxelles, Brussels) and was used for monitoring expression of mutant proteins. Maltose-binding protein (MBP) and thioredoxin (Trx) genes also were cloned in pBAD24. The strains A307 and Pop6590 deleted for chromosomal trxA and malE genes were used for monitoring expression of Trx and MBP mutants. Pop6590 was kindly provided by M. Hofnung (Institut Pasteur, Paris), and A307 was received from the E. coli Genetic Stock Center at Yale University. Mutagenesis. Thirty-nucleotide-long primers to generate CcdB mutants were designed by using OLIGO (Version 6.0, Molecular Biology Insights, Cascade, CO) and were obtained in 96-well format from the PAN Oligo facility at Stanford University. Each residue in CcdB was replaced with Ala, Asp, Glu, Arg, and Lys by using a mega-primer-based method of site-directed mutagenesis. The first PCR reaction was carried out in 96-well format by using PCR strips (each having eight tubes). Each tube contained a specific internal mutagenic primer and a vector-specific Conflict of interest statement: No conflicts declared.
doi:10.1073/pnas.0505089102 pmid:16251276 pmcid:PMC1283427 fatcat:obfq2phn3zb2zcfh6xqfem3ngu