RESUMEN
The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.
Asunto(s)
Repetición de Anquirina , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.
Asunto(s)
Sustitución de Aminoácidos/genética , Proteínas/química , Proteínas/genética , Repetición de Anquirina , Arginina/química , Arginina/genética , Arginina/metabolismo , Dicroismo Circular , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación/genética , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solubilidad , Relación Estructura-Actividad , TermodinámicaRESUMEN
The ankyrin repeat is one of the most common, modular, protein-protein interaction motifs in nature. To understand the structural determinants of this family of proteins and extract the consensus information that defines the architecture of this motif, we have designed a series of idealized ankyrin repeat proteins containing one, two, three, or four repeats by using statistical analysis of approximately 4,000 ankyrin repeat sequences from the PFAM database. Biophysical and x-ray crystallographic studies of the three and four repeat constructs (3ANK and 4ANK) to 1.26 and 1.5 A resolution, respectively, demonstrate that these proteins are well-folded, monomeric, display high thermostability, and adopt a very regular, tightly packed ankyrin repeat fold. Mapping the degree of amino acid conservation at each position on the 4ANK structure shows that most nonconserved residues are clustered on the surface of the molecule that has been designated as the binding site in naturally occurring ankyrin repeat proteins. Thus, the consensus amino acid sequence contains all information required to define the ankyrin repeat fold. Our results suggest that statistical analysis and the consensus sequence approach can be used as an effective method to design proteins with complex topologies. These generic ankyrin repeat proteins can serve as prototypes for dissecting the rules of molecular recognition mediated by ankyrin repeats and for engineering proteins with novel biological functions.
Asunto(s)
Repetición de Anquirina , Secuencia de Aminoácidos , Repetición de Anquirina/genética , Cromatografía en Gel , Secuencia de Consenso , Cristalografía por Rayos X , Evolución Molecular , Genes Sintéticos , Datos de Secuencia Molecular , Nefelometría y Turbidimetría , Resonancia Magnética Nuclear Biomolecular , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , SolucionesRESUMEN
Proteins containing stretches of repeating amino acid sequences are prevalent throughout nature, yet little is known about the general folding and assembly mechanisms of these systems. Here we propose myotrophin as a model system to study the folding of ankyrin repeat proteins. Myotrophin is folded over a large pH range and is soluble at high concentrations. Thermal and urea denaturation studies show that the protein displays cooperative two-state folding properties despite its modular nature. Taken together with previous studies on other ankyrin repeat proteins, our data suggest that the two-state folding pathway may be characteristic of ankyrin repeat proteins and other integrated alpha-helical repeat proteins in general.