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A Musacchio

Publications and source records attributed to A Musacchio.

49 records · Page 3Linked to original sources
PubMed1994

High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides.

Src-homology 3 (SH3) domains bind to proline-rich motifs in target proteins. We have determined high-resolution crystal structures of the complexes between the SH3 domains of Abl and Fyn tyrosine kinases, and two ten-residue proline-rich peptides derived from the SH3-binding proteins 3BP-1 and 3BP-2. The X-ray data show that the basic mode of binding of both proline-rich peptides is the same. Peptides are bound over their entire length and interact with three major sites on the SH3 molecules by both hydrogen-bonding and van der Waals contacts. Residues 4-10 of the peptide adopt the conformation of a left-handed polyproline helix type II. Binding of the proline at position 2 requires a kink at the non-proline position 3.

Amino Acid Sequence↗
PubMed1993

Crystallization and preliminary X-ray analysis of the periplasmic fragment of CyoA-a subunit of the Escherichia coli cytochrome o complex.

CyoA, an integral membrane protein, is a subunit of the Escherichia coli cytochrome o quinol oxidase complex. The C-terminal periplasmic domain of CyoA has been expressed in E. coli, purified and crystallized. Crystals were grown using ammonium sulphate as a precipitant. They have space group I222 or I2(1)2(1)2(1) and diffract X-rays to 2.3 A resolution.

Crystallization↗
PubMed1993

The PH domain: a common piece in the structural patchwork of signalling proteins.

The 'pleckstrin homology' domain is an approximately 100-residue protein module that has recently been added to the domain catalogue of signalling proteins. For this review we have made an extensive database search using a profile search method, and found a number of additional proteins that may contain PH domains. The PH domain is present in many kinases, isoforms of phospholipase C, GTPases, GTPase-activating proteins and nucleotide-exchange factors, including such proteins as Vav, Dbl and Bcr, and there are two PH domains in a guanine-nucleotide releasing factor of Ras. Many PH-domain-containing proteins interact with GTP-binding proteins. We have also identified a PH domain in beta-adrenergic receptor kinase exactly in the region that has already been shown to be involved in binding to the beta and gamma subunits of a heterotrimeric G protein. This suggests that PH domains may be involved in interactions with GTP-binding proteins.

Amino Acid Sequence↗
PubMed1993

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.

Bacterial Proteins↗
PubMed1993

Some effects of tonal fatiguing on spontaneous and distortion-product otoacoustic emissions.

Auditory fatiguing can be considered a suitable test to assess some cochlear mechanisms and diseases otherwise not easily detectable. Since spontaneous otoacoustic emissions (SOAEs) originate from active sources within the cochlea they show sensitive and early vulnerability to noise, displaying informative time-courses after overstimulation in the short (0-6 s) and in the long term (1-10 min) depending on the frequency of the fatiguing stimulus. Distortion product otoacoustic emissions too, in subjects with SOAEs, show interesting modifications after pure-tone exposure, detectable either on distortion product audiograms or in the growth functions. The modifications take place within a period of 5-7 minutes and strongly depend on the frequency of the fatiguing stimulus and on the closeness between SOAE and distortion product place. The data suggest that not only the interaction place between f1 and f2 has to be considered from a biomechanical and clinical point of view, but also the specific distortion product place on the cochlear partition.

Acoustic Stimulation↗
PubMed1992

Crystal structure of a Src-homology 3 (SH3) domain.

The Src-homologous SH3 domain is a small domain present in a large number of proteins that are involved in signal transduction, such as the Src protein tyrosine kinase, or in membrane-cytoskeleton interactions, but the function of SH3 is still unknown (reviewed in refs 1-3). Here we report the three-dimensional structure at 1.8 A resolution of the SH3 domain of the cytoskeletal protein spectrin expressed in Escherichia coli. The domain is a compact beta-barrel made of five antiparallel beta-strands. The amino acids that are conserved in the SH3 sequences are located close to each other on one side of the molecule. This surface is rich in aromatic and carboxylic amino acids, and is distal to the region of the molecule where the N and C termini reside and where SH3 inserts into the alpha-spectrin chain. We suggest that a protein ligand binds to this conserved surface of SH3.

Amino Acid Sequence↗
PubMed1992

SH3--an abundant protein domain in search of a function.

Src-homology 3 is a small protein domain of about 60 amino acid residues. It is probably made of beta-sheets. SH3 is present in a large number of eukaryotic proteins which are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. Here we review its occurrence and discuss possible functions of this domain.

Amino Acid Sequence↗
PubMed1992

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

Amino Acid Sequence↗
PubMed1991

Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector.

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

Amino Acid Sequence↗
PubMedDate not supplied

Recombinant Opc meningococcal protein, folded in vitro, elicits bactericidal antibodies after immunization.

The meningococcal Opc protein has been expressed as inclusion bodies in Escherichia coli. After cell disruption and successive washing of the insoluble fraction, insoluble proteins were solubilized in presence of the chaotropic agent guanidium hydrochloride. The extract was applied to a Reverse Phase High Performance Liquid Chromatography (RP-HPLC)-C4 column, for further purification. The obtained recombinant Opc protein was refolded in vitro, by the addition of several compounds to the resuspended solution. Over time, the progress of renaturation was tested by immunoblot with the human monoclonal antibody LuNm03 against the meningococcal Opc protein. LuNm03 recognizes a conformational epitope on the native meningococcal Opc protein. Having established the optimal conditions of renaturation. Balb/c mice were immunized to study the humoral immune response. The human at immune response elicited in mice was measured by ELISA and immunoblot, while the functional activity of these antibodies was assayed in a bactericidal test. According to our results, it was possible to obtain a recombinant Opc protein folded in vitro, with a conformation suitable enough to generate functional antibodies in mice, capable of killing meningococci in the presence of human complement.

Animals↗
PubMedDate not supplied

Suppression tuning characteristics of 2f1-f2 distortion product otoacoustic emissions.

The 2f1-f2 distortion product otoacoustic emission (DPOAE) is suppressed by a third tone following a frequency selective pattern and outlining tuning curves which are generally similar to neural and psychophysical findings. The most effective suppressor tone lies between f1 and f2; less suppressive effects are produced by an added tone that lies in the 2f1-f2 site. The slope of iso-suppression curves is much steeper on the higher flank than on the lower flank of the suppression curve (respectively 100-115 dB/octave and 25-35 dB/octave). A suppression fine structure can be observed in the region of the tip especially for higher DPs probably connected with the fine structure of distortion products and with the instability of cochlear activity. A saturation point, around 70 dB or more has been evidenced on the growth rate functions. Continuous and interrupted suppressor presentation can induce some differences in the responses. Suppression of DPOAEs can provide a wider knowledge on active non-linear mechanisms in the cochlea, and on frequency selectivity also in a clinical context.

Acoustic Stimulation↗
PubMedDate not supplied

[Distortion products in Menière's disease].

Distortion Product Otoacoustic Emissions (DPOAEs) are elicited by simultaneous application of two tones to the ear canal. These emissions are a result of nonlinear mechanical characteristics of the cochlea at a specific point and have a precise mathematical relation with the frequencies of the two eliciting primary tones f1 and f2. This frequency-selective property of DPOAEs suggests that they should be regarded as useful monitor of localized cochlear function at any predetermined frequency. The present study was designed to investigate DPOAEs in patients with Menière's disease. DPOAEs were recorded before and after glycerol administration to verify if the fluctuation of the hearing threshold induced by this hyperosmotic agent causes selective changes in the activity of the outer hair cells. DPOAEs were present in patients in which the mean duration of the disease was shorter compared to those patients without measurable DPOAEs. An improvement of DPOAEs after glycerol intatte was observed in a half of our cases. The improvement of DPOAEs does not appear to be brought about by modifications of the auditory threshold induced by glycerol.

Audiometry↗