Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry.

We have analyzed a newly described archaeal GimC/prefoldin homologue, termed MtGimC, by using nanoflow electrospray coupled with time-of-flight MS. The molecular weight of the complex from Methanobacterium thermoautotrophicum corresponds to a well-defined hexamer of two alpha subunits and four beta subunits. Dissociation of the complex within the gas phase reveals a quaternary arrangement of two central subunits, both alpha, and four peripheral beta subunits. By constructing a thermally controlled nanoflow device, we have monitored the thermal stability of the complex by MS. The results of these experiments demonstrate that a significant proportion of the MtGimC hexamer remains intact under low-salt conditions at elevated temperatures. This finding is supported by data from CD spectroscopy, which show that at physiological salt concentrations, the complex remains stable at temperatures above 65 degrees C. Mass spectrometric methods were developed to monitor in real time the assembly of the MtGimC hexamer from its component subunits. By using this methodology, the mass spectra recorded throughout the time course of the experiment showed the absence of any significantly populated intermediates, demonstrating that the assembly process is highly cooperative. Taken together, these data show that the complex is stable under the elevated temperatures that are appropriate for its hyperthermophile host and demonstrate that the assembly pathway leads exclusively to the hexamer, which is likely to be a structural unit in vivo.

Competitive binding to the oligopeptide binding protein, OppA: in-trap cleanup in an Fourier transform ion cyclotron resonance mass spectrometer.

This communication demonstrates that gentle infrared laser heating can remove unwanted buffer adducts from a gas-phase protein complex without dissociating the complex itself. Specifically, noncovalent complexes of the oligopeptide-binding protein, OppA, bound to either (Ala)3 or LysTrpLys were electrosprayed from aqueous buffer solution into a 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer. In addition to the intact complexes, several additional buffer adduct species were produced under the conditions of the experiment. Irradiation of the trapped ion population with a continuous-wave infrared CO2 laser at relatively low power (2.5 W) for 1 s dissociated the buffer adducts but retained the intact protein:peptide complexes. Adduct-free complex(es) were then readily identified, and signal-to-noise ratio also increased by an order of magnitude because the same number of protein ions are distributed over fewer species. Higher IR power (5 W for 1 s) dissociated the adduct-free complex(es) without internal fragmentation. The present in-trap clean-up technique may prove especially useful for identifying and screening the combinatorial library ligands most strongly bound to a receptor in the gas phase.

Mass spectrometry reveals elastase inhibitors from the reactive centre loop of alpha1-antitrypsin.

Peptides derived from the reactive centre loop of alpha1-antitrypsin, a serpin, were screened as potential elastase inhibitors by mass spectrometry. An octapeptide, MFLEAIPM, formed a 'stable' ternary complex with porcine elastase: one MFLEAIPM molecule reacted covalently with loss of water, whilst an additional peptide was bound non-covalently. Kinetic analyses suggested that MFLEAIPM may act as an uncompetitive inhibitor and that the activity was associated with the four N-terminal residues.

Detection and selective dissociation of intact ribosomes in a mass spectrometer.

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg(2+) concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 +/- 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.

Specificity and interactions of the protein OppA: partitioning solvent binding effects using mass spectrometry.

Mass spectrometry (MS) was used to characterise the binding of the 58 kDa protein OppA to 11 peptides with diverse properties. Peptides with two, three and five amino acid residues were added to OppA, and the mass spectra showed that the highest-affinity complexes are formed between OppA and tripeptide ligands. Lower-affinity complexes were observed for OppA and dipeptide ligands, and no complex formation was detected with pentapeptides or a tripeptide in which the N-terminal amino group was acetylated. Tripeptides containing a single d amino acid residue were found not to bind to native OppA. Evidence from the peak width and the, charge in the spectra of the complexes suggests that the bound peptides are encapsulated by the protein in a solvent-filled cavity in the gas phase of the mass spectrometer. Analysis of the proportions of peptide-bound and free proteins under low-energy MS conditions shows a good correlation with solution-phase K(d) measurements where available. Increasing the internal energy of the gas-phase complex led to dissociation of the complex. The ease of dissociation is interpreted in terms of the intrinsic stability of the complex in the absence of the stabilising effects of bulk solvent. The results from this study demonstrate insensitivity to the hydrophobic and ionic properties, of the side-chains of the peptides, in contrast to the investigation of other protein ligand systems by MS. Moreover, these findings are in accord with the physiological role of this protein in allowing into the cell di- and tripeptides containing naturally occurring amino acids, regardless of their sequence, while barring access to potentially harmful peptide mimics.

Disassembly of intact multiprotein complexes in the gas phase.

The observation of multiprotein complexes by mass spectrometry formerly relied upon chemical cross-linking to maintain interactions. Recent technological developments have enabled the observation of intact macromolecular complexes without modification. These assemblies, with masses far in excess of those measured previously, can be examined through controlled dissociation in the mass spectrometer, revealing information about their subunit interactions and topology.

Antifertility activity of Ricinus communis seed in female guinea pigs.

OBJECTIVE: To investigate the anti-fertility effect of Ricinus communis seed extract. DESIGN: Laboratory-based experiment. SETTING: Laboratory of the Department of Pharmacology, Faculty of Medicine, Addis Ababa University, Addis Ababa, Ethiopia in 1996. RESULTS: The seed extract was found to possess anti-implantation and abortifacient effects. It was also observed that the seed extract prolonged the oestrus cycle of guinea pigs. The dioestrus phase was significantly prolonged as well. After stopping administering the extract, however, the normal dioestrus phase and oestrus cycle started to resume. The seed extract also reduced the weight of the uterus without affecting that of the ovaries significantly. CONCLUSION: Ricinus communis possesses an anti-fertility effect in female guinea pigs, which might be extrapolated in human beings. These findings might support the accredited claim of its traditional use to avoid unwanted pregnancies. Further studies, however, should be pursued.

Dissection of multi-protein complexes using mass spectrometry: subunit interactions in transthyretin and retinol-binding protein complexes.

Complexes formed between transthyretin and retinol-binding protein prevent loss of retinol from the body through glomerular filtration. The interactions between these proteins have been examined by electrospray ionization combined with time-of-flight mass analysis. Conditions were found whereby complexes of these proteins, containing from four to six protein molecules with up to two ligands, are preserved in the gas phase. Analysis of the mass spectra of these multimeric species gives the overall stoichiometry of the protein subunits and provides estimates for solution dissociation constants of 1.9 +/- 1.0 x 10(-7) M for the first and 3.5 +/- 1.0 x 10(-5) M for the second retinol-binding protein molecule bound to a transthyretin tetramer. Dissociation of these protein assemblies within the gas phase of the mass spectrometer shows that each retinol-binding protein molecule interacts with three transthyretin molecules. Mass spectral analysis illustrates not only a correlation with solution behavior and crystallographic data of a closely related protein complex but also exemplifies a general method for analysis of multi-protein assemblies.