Problems Affecting Labor

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by ℙ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G A (a 3-α helix fold) and G B (an α/β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G A domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G Bs, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey

La mioglobina: ingegneria proteica ed analisi della struttura genica

Dottorato di ricerca in biochimica. 7. ciclo. A.a. 1991-94. Docente guida e Coordinatore Maurizio BrunoriConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Aplysia limacina myoglobin cDNA cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis.

The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back transplanting the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)-containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved