RESUMEN
Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA. In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper. We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper. DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains. These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Leucina Zippers/genética , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Secuencia Conservada/genética , Proteínas de Unión al ADN/síntesis química , Dimerización , Escherichia coli/genética , Proteínas Fúngicas/síntesis química , Factores de Unión a la G-Box , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/síntesis química , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/síntesis química , Secuencias Repetitivas de Aminoácido/genética , Termodinámica , Factores de Transcripción/síntesis químicaRESUMEN
bZip transcription factors contain two regions that are required for DNA binding: a leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The spacing between these subdomains is strictly conserved, and changes in this spacing result in a loss of function. Using an in vitro selection strategy, we have investigated the ability of a bZip protein with incorrect spacing between these two regions to bind specifically to DNA. Surprisingly, we find that although such a protein does not bind to its predicted site, it is possible to isolate a pool of DNAs that bind with very similar affinity to that of GCN4 for its optimum DNA site.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Proteínas de Unión al ADN/química , Ensayo de Cambio de Movilidad Electroforética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Factores de Unión a la G-Box , Leucina Zippers , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/metabolismo , Fragmentos de Péptidos , Unión Proteica , Conformación Proteica , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/química , LevadurasRESUMEN
Recent structural studies have highlighted the importance of antiparallel coiled coils in nature. In addition, well-behaved, model antiparallel coiled coils have been designed and used for the reassembly of protein fragments and for the study of the energetic contributions of various interactions to helix orientation specificity. Finally, high-resolution structural data are available for designed helical bundles, allowing an evaluation of the success of state-of-the-art protein design efforts.
Asunto(s)
Conformación Proteica , Algoritmos , Animales , Proteínas Bacterianas/química , Bases de Datos Factuales , Predicción , Antígenos de la Hepatitis/química , Antígenos de Hepatitis delta , Humanos , Modelos Moleculares , Proteína Quinasa C , Proteínas Serina-Treonina Quinasas/química , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas de Unión al ARN/química , Serina-ARNt Ligasa/química , Espectrina/químicaRESUMEN
PURPOSE/OBJECTIVES: To describe symptoms associated with radiation therapy necessary to develop preparatory concrete, objective information for women with cervical or uterine cancer. DESIGN: Prospective, descriptive. SETTING: University-affiliated radiation oncology department in the mid-south. SAMPLE: 49 of 52 women undergoing external beam radiation and low-dose rate brachytherapy for cervical or uterine cancer. Stage of disease ranged from I-IIIA. Mean age of subjects was 54 years; mean years of education was 11. METHODS: Investigator-developed symptom checklist based on prior research. MAIN RESEARCH VARIABLES: Symptom reports. FINDINGS: At least 40% of the women reported difficulty sleeping, fatigue, diarrhea, anorexia, nausea, urinary frequency, dysuria, vaginal discharge, and perineal irritation. Incidence and timing of symptoms varied by operative status and brachytherapy timing. CONCLUSIONS: Findings confirm and clarify the symptoms women associate with radiation treatment for cervical or uterine cancer. IMPLICATIONS FOR NURSING PRACTICE: Assessing research findings for relevance to the care of patients in specific settings is necessary. These findings are appropriate for developing preparatory information for women undergoing external beam radiation and low-dose rate brachytherapy.
Asunto(s)
Braquiterapia/efectos adversos , Educación del Paciente como Asunto , Neoplasias del Cuello Uterino/radioterapia , Neoplasias Uterinas/radioterapia , Adulto , Anciano , Diarrea/etiología , Fatiga/etiología , Femenino , Humanos , Persona de Mediana Edad , Náusea/etiología , Estudios Prospectivos , Trastornos del Sueño-Vigilia/etiología , Trastornos Urinarios/etiología , Excreción Vaginal/etiologíaRESUMEN
Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. The factors that determine a preference for a given relative helix orientation are incompletely understood. The helix orientation preference for the designed coiled coil, Acid-a1-Base-a1, was measured previously. This model system therefore provides a means for the experimental determination of the energetic contribution of a variety of interactions to helix orientation specificity. The antiparallel preference for Acid-a1-Base-a1 is imparted by a single buried polar interaction. Interhelical Coulombic interactions between residues at the e and g positions have been proposed to influence helix orientation preference. In the Acid-a1-Base-a1 heterodimer, potentially attractive Coulombic interactions are expected in both orientations. To determine the energetic consequences of Coulombic interactions for helix orientation preference, we have positioned a single charged residue in each peptide such that exclusively favorable interhelical Coulombic interactions can occur only in the parallel orientation. In contrast, two potentially repulsive interactions are expected in the antiparallel orientation. Because the buried polar interaction can occur only in the antiparallel orientation, interhelical Coulombic interactions favor the parallel orientation and the potential to form a buried polar interaction favors the antiparallel orientation. We find no clear preference for an antiparallel orientation in the resulting heterodimer, Acid-Ke-Base-Eg, suggesting that interhelical Coulombic interactions and a buried polar interaction are of approximately equal importance for helix orientation specificity. Stability measurements indicate that maintenance of all favorable electrostatic interactions and/or avoidance of two potentially repulsive interactions contributes approximately 2.1 kcal/mol to helix orientation preference.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Dimerización , Disulfuros/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Desnaturalización Proteica/efectos de los fármacos , Ingeniería de Proteínas , Estructura Secundaria de Proteína/efectos de los fármacos , Electricidad Estática , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismo , Temperatura , Termodinámica , Ultracentrifugación , Urea/farmacologíaRESUMEN
bZip proteins contain a bipartite DNA-binding motif consisting of a "leucine zipper" dimerization domain and a highly charged "basic region" that directly contacts DNA. These transcription factors form dimeric complexes with each monomer recognizing half of a symmetric or nearly symmetric DNA site. We have found that the bZip protein GCN4 can also bind with high affinity to DNA sites containing only a single GCN4 consensus half-site. Because several recent lines of evidence have suggested a role for monomeric DNA binding by bZip proteins, we investigated the structure of the GCN4.half-site complex. Quantitative DNA binding and affinity cleaving studies support a model in which GCN4 binds as a dimer, with one monomer making specific contacts to the consensus half-site and the other monomer forming nonspecific contacts that are nonetheless important for binding affinity. We also examined the folding transition induced in the basic regions of this complex upon binding DNA. Circular dichroism (CD) studies demonstrate that the basic regions of both monomers are helical, suggesting that a protein folding transition may be required for both specific and nonspecific DNA binding by GCN4.
Asunto(s)
ADN/química , ADN/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Ácido Edético/química , Cinética , Leucina Zippers , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Coiled coils consist of bundles of two or more alpha-helices that are aligned in a parallel or an antiparallel relative orientation. The designed peptides, Acid-p1 and Base-p1, associate in solution to form a parallel, heterodimeric two-stranded coiled coil [O'Shea, E. K., Lumb, K. J., and Kim, P. S. (1993) Curr. Biol. 3, 658]. The buried interface of this complex is formed by hydrophobic Leu residues, with the exception of an Asn residue from each strand that is positioned to engage in a buried polar interaction. Substitution of these buried Asn residues by Leu residues results in a loss of structural uniqueness, as evidenced by a lack of a particular helix orientation in the Acid-Base coiled-coil complex [Lumb, K. J., and Kim, P. S. (1995) Biochemistry 34, 8642]. Here, we alter the positions of the Asn residues in the Acid and Base peptides such that a buried polar interaction is only expected to occur when the helices are in an antiparallel orientation. The resulting peptides, Acid-a1 and Base-a1, associate to form a helical heterodimer, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. The helix orientation preference has been measured using covalently linked, disulfide-containing heterodimers in which the constituent peptides are constrained to interact in either a parallel or an antiparallel orientation. Although both the parallel and antiparallel heterodimers form stable, helical structures, the antiparallel heterodimer is the predominant species at equilibrium when the heterodimers are allowed to undergo thiol-disulfide exchange. In addition, the antiparallel heterodimer is more stable to chemical denaturation than the parallel counterpart by approximately 2.3 kcal/mol. These results demonstrate that a single buried polar interaction in the interface between the helices of a coiled coil is sufficient to determine the relative orientation of its constituent helices.
Asunto(s)
Péptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Dicroismo Circular , Dimerización , Disulfuros/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos/síntesis química , Compuestos de Sulfhidrilo/químicaAsunto(s)
Infecciones por Haemophilus/prevención & control , Haemophilus influenzae tipo b , Rifampin/uso terapéutico , Cápsulas Bacterianas , Femenino , Infecciones por Haemophilus/transmisión , Vacunas contra Haemophilus , Humanos , Lactante , Polisacáridos Bacterianos , Práctica de Salud Pública , RecurrenciaRESUMEN
The alpha-helices of coiled-coil proteins are predominantly parallel, in contrast to the general preference for an antiparallel orientation of interacting alpha-helices found in globular proteins. One intriguing exception is the antiparallel, two-stranded coiled coil comprising the long helical arm of the bacterial seryl tRNA synthetases (SRS). A recombinant 82-residue peptide corresponding to the helical arm of Escherichia coli SRS folds into a stable, monomeric, helical structure in the absence of the rest of the protein, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. However, peptides corresponding to the individual helices of SRS are unstructured at neutral pH and do not associate appreciably at total peptide concentrations up to 100 microM. Covalent attachment of the the two peptides through a nonnatural, disulfide-containing linker restores structure and allows study of variants in which the individual helices are constrained to interact in either an antiparallel or a parallel orientation. We find that the antiparallel species are substantially more helical and more stable to thermal denaturation than their parallel counterpart. Thus, the SRS helical arm is an autonomously folding unit, and, unlike most other coiled coils, has an intrinsic preference for an antiparallel orientation of its constituent helices.
Asunto(s)
Escherichia coli/enzimología , Estructura Secundaria de Proteína , ARN de Transferencia de Serina/química , Serina-ARNt Ligasa/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Pliegue de ProteínaRESUMEN
BACKGROUND: The transcription factor AP-1 activates the expression of numerous genes in response to mitogenic stimuli. AP-1 regulates gene expression both through solitary binding to independent recognition sites and, in cooperation with various heterologous transcription factors, through targeting to composite response elements. The two subunits that make up the AP-1 heterodimer, Fos and Jun, possess identical residues at positions that make sequence-specific contacts to DNA. This degeneracy leaves the protein with no apparent way of orienting itself uniquely on DNA by differentially recognizing its two non-identical half-sites. Here, we have analyzed the orientation of the AP-1 basic-leucine-zipper (bZip) domain on a cognate site, both alone and in the cooperative complex formed together with the 'nuclear factor of activated T cells' (NFATp). RESULTS: The results of affinity cleaving experiments demonstrate that, in solution, the AP-1 bZip binds DNA as a mixture of two orientational isomers. However, in the cooperative complex formed with NFATp on a composite response element, the AP-1 bZip adopts a single orientation, with Jun and Fos bound to the NFATp-proximal and NFATp-distal half-sites, respectively. Protein cross-linking experiments demonstrate that protein-protein contacts are responsible for this 'orientational locking'. CONCLUSIONS: Our results demonstrate that, through protein-protein interactions, one protein can force another to adopt a single DNA-bound orientation. Thus, cooperative interactions between adjacent regulatory proteins can influence not only the energetics of their interactions with DNA, but also their precise geometric and stereochemical arrangement. Because orientational isomers present markedly different structures to the transcriptional apparatus, it seems likely that orientation will exert an effect on the ability to activate transcription.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Unión Proteica , Conformación ProteicaRESUMEN
The iron-binding and oxygen-activating domain of the natural product bleomycin [pyrimidoblamic acid-beta-hydroxy-L-histidine (PBA-beta-OH-His)] was attached to the NH2 terminus of the DNA binding domain of Hin recombinase (residues 139-190). This hybrid 54-residue protein PBA-beta-OH-His-Hin-(139-190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol, Fe(II).PBA-beta-OH-His-Hin-(139-190) cleaves DNA with specificity remarkably similar to that of Fe(II).EDTA-Hin(139-190). Analysis of the cleavage patterns suggests that site-specific DNA cleavage is mediated by a localized diffusible species, in contrast with cleavage by bleomycin, which occurs through a nondiffusible oxidant. This has implications for the design of second-generation artificial sequence specific DNA cleaving proteins and defines limitations in current efforts to create atom-specific chemistry on DNA.
Asunto(s)
Bleomicina/síntesis química , ADN Nucleotidiltransferasas/síntesis química , Fragmentos de Péptidos/síntesis química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Bleomicina/química , Bleomicina/metabolismo , ADN/genética , ADN/metabolismo , ADN Nucleotidiltransferasas/química , ADN Nucleotidiltransferasas/metabolismo , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Especificidad por SustratoRESUMEN
Affinity cleaving proteins have been synthesized based on the DNA-binding domain of the yeast transcriptional activator GCN4 with the DNA cleaving moiety Fe.EDTA attached at the NH2 terminus [Oakley, M. G., & Dervan, P. B. (1990) Science 248, 847]. Cleavage patterns generated by Fe-EDTA-GCN4(226-281) bound to the DNA sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3' reveal that the NH2 termini of the GCN4 DNA-binding domain are located in the major groove of DNA, 9-10 base pairs apart, consistent with a Y-shaped dimeric structure. 1-Methylimidazole-2-carboxamide netropsin (2-ImN) is a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer [Mrksich, M., Wade, W. S., Dwyer, T. J., Geierstanger, B. H., Wemmer, D.E., & Dervan, P. B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7586]. Through the use of Fe.EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4-(226-281) and the dimeric peptide 2-ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe.EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Autorradiografía , Secuencia de Bases , ADN/química , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Especificidad por SustratoRESUMEN
The NH2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 have been mapped on the binding sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3'. Affinity cleaving was effected by synthetic GCN4 proteins with Fe.EDTA moieties at the NH2-terminus. Analysis of the DNA cleavage patterns for dimers of the Fe.EDTA-proteins corresponding to GCN4 residues 222 to 281 and 226 to 281 revealed that the NH2-termini were in the major groove nine to ten base pairs apart and were symmetrically displaced four to five base pairs from the central C of the recognition site. This result is consistent with the Y-shaped scissor grip-leucine zipper model recently proposed for a class of DNA binding proteins important in the regulation of gene expression.