RESUMO
A method of combinatorial cassette mutagenesis was designed to readily determine the informational content of individual residues in protein sequences. The technique consists of simultaneously randomizing two or three positions by oligonucleotide cassette mutagenesis, selecting for functional protein, and then sequencing to determine the spectrum of allowable substitutions at each position. Repeated application of this method to the dimer interface of the DNA-binding domain of lambda repressor reveals that the number and type of substitutions allowed at each position are extremely variable. At some positions only one or two residues are functionally acceptable; at other positions a wide range of residues and residue types are tolerated. The number of substitutions allowed at each position roughly correlates with the solvent accessibility of the wild-type side chain.
Assuntos
Proteínas de Ligação a DNA , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sítios de Ligação , Códon , DNA/genética , DNA/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Plasmídeos , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
lambda Cro is a dimeric DNA binding protein. Random mutagenesis and a selection for Cro activity have been used to identify the contacts between Cro subunits that are crucial for maintenance of a stably folded structure. To obtain equivalent contacts in a monomeric system, a Cro variant was designed and constructed in which the antiparallel beta-ribbon that forms the dimer interface was replaced by a beta-hairpin. The engineered monomer has a folded structure similar to wild type, is significantly more stable than wild type, and exhibits novel half-operator binding activity.
Assuntos
Bacteriófago lambda/genética , Proteínas de Ligação a DNA , Variação Genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/genética , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Proteínas Repressoras/metabolismo , Termodinâmica , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain. This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease. Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP. Cells with an sspB mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Oligopeptídeos/metabolismo , Serina Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Endopeptidase Clp , Escherichia coli/enzimologia , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Óperon , Ribossomos/metabolismo , Especificidade por SubstratoRESUMO
Variants of lambda repressor and cytochrome b562 translated from messenger RNAs without stop codons were modified by carboxyl terminal addition of an ssrA-encoded peptide tag and subsequently degraded by carboxyl terminal-specific proteases present in both the cytoplasm and periplasm of Escherichia coli. The tag appears to be added to the carboxyl terminus of the nascent polypeptide chain by cotranslational switching of the ribosome from the damaged messenger RNA to ssrA RNA.
Assuntos
Grupo dos Citocromos b/metabolismo , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Processamento de Proteína Pós-Traducional , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Alanina , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon de Terminação , Grupo dos Citocromos b/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
A "switch" mutant of the Arc repressor homodimer was constructed by interchanging the sequence positions of a hydrophobic core residue, leucine 12, and an adjacent surface polar residue, asparagine 11, in each strand of an intersubunit beta sheet. The mutant protein adopts a fold in which each beta strand is replaced by a right-handed helix and side chains in this region undergo significant repacking. The observed structural changes allow the protein to maintain solvent exposure of polar side chains and optimal burial of hydrophobic side chains. These results suggest that new protein folds can evolve from existing folds without drastic or large-scale mutagenesis.
Assuntos
Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Proteínas Virais/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Asparagina/química , Dicroísmo Circular , Ligação de Hidrogênio , Leucina/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Virais Reguladoras e AcessóriasRESUMO
An amino acid sequence encodes a message that determines the shape and function of a protein. This message is highly degenerate in that many different sequences can code for proteins with essentially the same structure and activity. Comparison of different sequences with similar messages can reveal key features of the code and improve understanding of how a protein folds and how it performs its function.
Assuntos
Sequência de Aminoácidos , Proteínas de Ligação a DNA , Proteínas/fisiologia , Gráficos por Computador , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas/ultraestrutura , Proteínas Repressoras , Relação Estrutura-Atividade , Propriedades de Superfície , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.
Assuntos
Bacteriófago lambda/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Zíper de Leucina/genética , Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Sequência de Aminoácidos , Variação Genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Conformação Proteica , Distribuição Aleatória , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Protein design efforts are beginning to yield molecules with many of the properties of natural proteins. Such experiments are informed by and contribute to our understanding of the sequence determinants of protein folding and stability. The most important design elements seem to be the proper placement of hydrophobic residues along the polypeptide chain and the ability of these residues to form a well packed core. Buried polar interactions, turn and capping motifs and secondary structural propensities also contribute, although probably to a lesser extent.
Assuntos
Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Sequência de Aminoácidos , Fenômenos Químicos , Físico-Química , Desenho de Fármacos , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Crystal and cocrystal structures of the LacI and PurR repressors reveal a novel use of hinge alpha helices which bind in the minor groove of the operator and mediate transmission of the allosteric signals that modulate DNA-binding activity.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Proteínas Repressoras/química , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Cristalografia por Raios X , Óperon Lac , Repressores Lac , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Repressoras/fisiologia , Transcrição GênicaRESUMO
BACKGROUND: The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain-DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues. RESULTS: The crystal structure of a complex containing the engrailed Gln50-->Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 A resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4. CONCLUSIONS: The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain-DNA recognition and further clarifies the roles of water molecules near residues 50 and 51.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Lisina/química , Fatores de Transcrição , Animais , Asparagina/química , Cristalografia por Raios X , Drosophila/química , Ligação de Hidrogênio , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
RecA-mediated cleavage of the bacteriophage lambda repressor results in inactivation of the protein and leads to induction of the lambda prophage. Here, we report the identification of three mutations in lambda repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the ind- mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.
Assuntos
Bacteriófago lambda/metabolismo , Recombinases Rec A/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Cinética , Mutação , Proteínas Repressoras/isolamento & purificação , Compostos de Sulfidrila/metabolismo , Supressão Genética , Proteínas Virais/isolamento & purificaçãoRESUMO
Cassette mutagenesis has been used to investigate how internal packing interactions help to specify a protein's three-dimensional structure and stability. Three interacting residues in the hydrophobic core of the N-terminal domain of lambda repressor were randomized combinatorially. The randomization was restricted to the five amino acids Val, Leu, Ile, Met and Phe, thereby generating a sterically diverse set of core sequences composed solely of hydrophobic residues. We have isolated 78 of the 125 possible sequences generated by this randomization. Approximately 70% of the isolated sequences show some level of biological activity, and thus still carry sufficient information to encode the basic structure of lambda repressor. An assay based on the temperature dependence of activity in vivo has been used to estimate the relative activities and thermal stabilities of the set of mutants. In addition, nine mutants have been purified and their stabilities and DNA binding activities characterized in vitro. Of the 56 active sequences, only two, in addition to the wild-type, maintain the wild-type level of stability and activity. All three of these proteins satisfy stringent requirements for specifically shaped residues at each position. All of the remaining active sequences have reduced stabilities and/or reduced DNA binding affinities. These and previous results suggest that there are two levels of structural information encoded in core residues. At the first level, the basic structural information appears to reside largely in the hydrophobic character of these residues. The majority of sequences that simply maintain hydrophobicity at core positions are able to adopt the overall lambda repressor fold and maintain moderate stability. At the second, more detailed level, specific steric features of these residues and their packing interactions clearly act as important determinants of the protein's precise structure and stability. These results imply that many of the basic structural features of a protein could be predicted from relatively simple, degenerate sequence patterns.
Assuntos
Proteínas de Ligação a DNA , Conformação Proteica , Proteínas/química , Proteínas Repressoras/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Estabilidade de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Sintéticos , Temperatura Alta , Modelos Moleculares , Mutagênese Insercional , Fenótipo , Plasmídeos , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Mapeamento por Restrição , Termodinâmica , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.
Assuntos
Bacteriófago lambda/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Bacteriófago lambda/imunologia , Sequência de Bases , DNA Viral , Proteínas de Ligação a DNA , Imunidade , Mutação , Proteínas Repressoras/metabolismo , Temperatura , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
We have described a set of mutations that alter side-chains on the operator binding surface of lambda repressor. In this paper, we study the interactions of 12 purified mutant repressors with operator and non-operator DNA. The mutant proteins have operator affinities that are reduced from tenfold to greater than 10,000-fold compared to wild-type. Nine of the mutants have affinities for non-operator DNA that are similar to wild-type, two mutants show decreased non-specific binding, and one mutant has increased affinity for non-operator DNA. We discuss these findings in terms of the structural and energetic contributions of side-chain--DNA interactions, and show that certain contacts between the repressor and the operator backbone contribute both energy and specificity to the interaction.
Assuntos
Bacteriófago lambda/genética , DNA Viral/genética , Proteínas de Ligação a DNA , Mutação , Regiões Operadoras Genéticas , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Gráficos por Computador , Simulação por Computador , Escherichia coli/genética , Metilação , Modelos Genéticos , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Under physiological conditions, lambda repressor can be inactivated in vivo or in vitro by RecA-mediated cleavage of the polypeptide chain. The repressor protein is thought to cleave itself, with RecA acting to stimulate autodigestion. ind- repressor mutants are resistant to RecA-mediated inactivation in vivo. In this paper, we report the purification of 15 ind- repressor proteins and the behaviors of these proteins in the RecA-mediated and autodigestion cleavage reactions. None of these proteins undergoes substantial RecA-dependent cleavage. However, eight mutant proteins autodigest at the same rate as wild-type repressor, six mutants do not autodigest or autodigest slower, and one mutant autodigests faster than wild-type. We discuss these results with respect to repressor structure and RecA-binding, and suggest possible roles for the RecA protein in the cleavage reaction.
Assuntos
Proteínas de Ligação a DNA , Recombinases Rec A/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , DNA Viral/metabolismo , Cinética , Mutação , Regiões Operadoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Amino acid changes known to be structurally allowed in Arc repressor were used to aid in the identification of proteins with sequence similarity to Arc and the related Mnt repressor. The sequences of the TraY proteins from the F episome and related episomes were found to be similar to those of Arc and Mnt.
Assuntos
Plasmídeos/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Software , Proteínas Virais Reguladoras e AcessóriasRESUMO
Arg2, in the N-terminal arm of the Mnt repressor, plays an important role in determining operator-binding specificity. In the complex of the Mnt tetramer with the 21 base-pair mnt operator, there are four potential sites for Arg2 interactions, two in the central region of the operator and two on the outer flanks of the operator. Single-chain variants of the dimeric N-terminal domain of Mnt containing one Arg2 residue and one Lys2 or Met2 residue were constructed and interactions with operator DNA were probed using Fe. EDTA affinity cleavage. The results of these orientation studies show that the majority of the energetically significant interactions mediated by Arg2 occur in the central region of the mnt operator. The RK2, RA2, and RM2 mutations reduce the free energy of operator binding by 1.7 kcal/mol, 3.3 kcal/mol, and 4.9 kcal/mol, respectively. Double-mutant thermodynamic cycle analyses using the RA2, RM2, and operator variants also reveal interaction free energies between Arg2 and operator base-pairs 9, 10, 11, 12 and 13, which in aggregate account for most of the Arg2 contribution to operator binding.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regiões Operadoras Genéticas/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Bases , Pegada de DNA , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Dimerização , Ácido Edético/metabolismo , Compostos Férricos/metabolismo , Genes Virais/genética , Modelos Moleculares , Mutação , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta/genética , Especificidade por Substrato , Termodinâmica , Proteínas Virais/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Dimers of P22 Arc repressor bind to half-sites of the 21 bp arc operator and interact cooperatively to stabilize a DNA-bound tetramer. Mutation of Ser35 (a residue in the dimer-dimer interface) to Arg or Leu disrupts cooperative binding. The mutant proteins have near wild-type stabilities, give operator footprints like wild-type, and prevent binding of RNA polymerase to the Pant promoter in vitro. These mutants are, however, largely inactive in vivo. Thus, although cooperativity is not structurally required for repression, it appears that the additional DNA-binding energy from dimer-dimer cooperativity is required for normal biological function. Altering the spacing between the DNA half-sites by even one base-pair eliminates dimer-dimer cooperativity, indicating that Arc dimers need to be oriented correctly by half-site binding to allow the interactions that stabilize the tetrameric complex.
Assuntos
Bacteriófago P22/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Bacteriófago P22/genética , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Mutação Puntual , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
By binding to adjacent subsites in its 21 base-pair operator, Arc represses transcription from two divergent promoters, Pant and Pmnt, in the immunity I operon of bacteriophage P22. Arc dimers bind to each subsite with nanomolar affinities and interact through protein-protein interactions to stabilize binding further. Here, we show that an Arc dimer bound to a single subsite reduces the rate of RNA polymerase open-complex formation and represses transcription from Pant and Pmnt promoter variants to varying degrees. Occupancy of the subsite proximal to the Pant-35 region results in significantly greater repression than occupancy of the- 10 proximal subsite. For repression of Pmnt, Arc bound at the- 10 proximal subsite is more effective than Arc bound at the- 35 proximal subsite. Because of the divergent orientations of the two promoters, the-35 proximal site in Pant is the same as the- 10 proximal site in Pmnt. Thus, in both cases, the same operator subsite is primarily responsible for repression of transcription initiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/análise , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Virais Reguladoras e AcessóriasRESUMO
We report the chemically determined sequence of most of the polypeptide chain of the coat protein of tomato bushy stunt virus. Peptide locations have been determined by comparison with the high-resolution electron density map from X-ray crystallographic analysis as well as by conventional chemical overlaps. Three small gaps remain in the 387-residue sequence. Positively charged side-chains are concentrated in the N-terminal part of the polypeptide (the R domain) as well as on inward-facing surfaces of the S domain. There is homology of S-domain sequences with structurally corresponding residues in southern bean mosaic virus.