RESUMEN
p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.
Asunto(s)
Guanosina Trifosfato/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas de Unión al GTP rac/química , Proteínas de Unión al GTP rac/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calorimetría , Guanosina Trifosfato/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Termodinámica , Proteína RCA2 de Unión a GTPRESUMEN
The core light-harvesting LH1 complex of Rhodospirillum rubrum consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll (BChl) a molecule. In this work, we describe a technique that allows the replacement of the natural, Mg BChl a cofactors present in this protein by Zn-bacteriopheophytin (Zn-Bpheo). This technique makes use of the well-characterized, reversible dissociation of LH1 induced by the detergent beta-octylglucoside. Incubation of partially dissociated LH1 with exogeneous pigments induces an equilibrium between the protein-bound BChl and the exogeneous pigment. This results in the binding of chemically modified pigments to LH1, in amounts which depend on the pigment composition and concentration of the exchange buffer. This method can yield information on the relative affinities of the LH1 protein-binding sites for the different pigments BChl and Zn-Bpheo and can also be used to obtain fully reassociated LH1 proteins, with a variable content of modified pigment, which may be precisely monitored. Absorption and FT-Raman spectroscopy indicate that this exchange procedure leads to LH1 proteins containing modified pigments, but retaining a binding site structure identical to that of native LH1. Furthermore, examination of the binding curves suggests that there are two distinguishable binding sites, probably corresponding to the two polypeptides, with very different properties. One of these two binding sites shows a marked preference for Zn-Bpheo over BChl, while the other binding site appears to prefer BChl.
Asunto(s)
Proteínas Bacterianas , Feofitinas/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodospirillum rubrum/química , Zinc/química , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Dicroismo Circular , Complejos de Proteína Captadores de Luz , Modelos Biológicos , Modelos Químicos , Feofitinas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , Rhodospirillum rubrum/metabolismo , Espectrofotometría , Espectrometría Raman , Relación Estructura-Actividad , Zinc/metabolismoRESUMEN
Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.
Asunto(s)
Proteínas Bacterianas , Bacterioclorofilas/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Membrana Púrpura/química , Complejos de Proteína Captadores de Luz , Conformación Proteica , Rhodobacter sphaeroides/química , Rhodopseudomonas/química , Rhodospirillum/química , Rhodospirillum rubrum/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Resonance Raman spectroscopy of an antenna protein from the brown alga Laminaria saccharina has been used to investigate the molecular structure of this light-harvesting complex (LHC) at the level of its bound pigments, chlorophylls (chl) a and c and the xanthophyll fucoxanthin. Evidence has been obtained for the conservation of pigment structure during the isolation procedure used. Six chl a and two chl c molecules are indicated from the positions and relative contributions of stretching modes of their keto-carbonyl groups. Of special interest is the presence of a population of chls a having a protein-binding conformation highly similar to that seen in antenna proteins from higher plants, possibly indicating a common structural motif within this extended gene family. The eight fucoxanthin molecules evidenced are all in the all-trans conformation; however, one or two have a highly twisted configuration. The results are discussed in terms of common and varying structural features of LHCs in higher plants and algae.