Contact Sites of Peptide–Oligoribonucleotide Cross-Links Identified by a Combination of Peptide and Nucleotide Sequencing with MALDI MS

We have investigated peptide–oligoribonucleotide complexes isolated from cross-linked Escherichia coli 30S ribosomal subunits in order to identify the contact sites of these complexes at the molecular level. For this purpose, reversed-phase (RP) HPLC-purified peptide–oligoribonucleotide complexes were submitted to N-terminal amino acid sequencing in order to determine the cross-linked peptide moiety and were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for calculation of the nucleotide composition of the cross-linked complex. Subsequently, for nucleotide sequence information the complexes were partially hydrolyzed or treated with exonucleases and analyzed again by MALDI-MS. Applying this technique, we were able to identify the cross-linked oligoribonucleotide parts in contact with distinct peptide regions derived from ribosomal proteins S4, S7, S8, and S17 from E. coli.     

Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

Matrix-assisted laser desorption–ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures

In theory, peptide mass fingerprinting by matrix assisted laser desorption–ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower μl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.     

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E--F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide–heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide–heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme–peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P₅₀ values. The P₅₀ values ranged from 3.8 mmHg for the heme–peptide B7 complex to 13.7 mmHg for the heme–peptide D13 complex (under the same conditions, P₅₀ values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.     

Binding of protein synthesis initiation factor IF1 to 30 S ribosomal subunits: effects of other initiation factors and identification of proteins near the binding site.

The effects of other components of the initiation complex on Escherichia coli initiation factor IFI binding to 30 S ribosomal subunits were studied. Binding of [¹⁴C]IF1 in the absence of other initiation complex components was slight. Addition of either IF2 or IF3 stimulated binding to a variable extent. Maximum binding was observed when both IF2 and IF3 were present. Addition of GTP, fMet-tRNA, and phage R17 RNA caused little or no further stimulation of [¹⁴C]IF1 binding. A maximum of 0.5 molecule of [¹⁴C]IF1 bound per 30 S subunit in the presence of an excess of each of the three factors over 30 S subunits.Complexes of 30 S subunits, [¹⁴C]IF1, IF2, and IF3 were treated with the bifunctional protein cross-linking reagent dimethyl suberimidate in order to identify the ribosomal proteins near the binding site for IF1. Non-cross-linked [¹⁴C]IF1 was removed from the complexes by sedimentation through buffer containing a high salt concentration, and total protein was extracted from the pelleted particles. Approximately 12% of the [¹⁴C]IF1 was recovered in the pellet fraction. The mixture of cross-linked products was analyzed by polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Autoradiography of the gel showed radioactive bands with molecular weights of 21,000, 25,000, and many greater than 120,000. The results indicate that [¹⁴C]IF1 was cross-linked directly to at least two ribosomal proteins. Analysis of the cross-linked mixture by radioimmunodiffusion with specific antisera prepared against each of the 30 S ribosomal proteins showed radioactivity in the precipitin bands formed with antisera against S12 and S19, and in lower yield with those against S1 and S13. Antiserum against IF2 also showed [¹⁴C]IF1 in the precipitin band. The results show that [¹⁴C]IF1 was present in covalently cross-linked complexes containing 30 S ribosomal proteins S1, S12, S13 and S19, and initiation factor IF2. The same ribosomal proteins have been implicated in the binding sites for IF2 and IF3. The results suggest that the three initiation factors bind to the 30 S subunit at the same or overlapping sites.     

Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit

Complexes of 30 S subunits and [¹⁴C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.     

Positioning of mRNA 3' of the a site bound codon on the human 80S ribosome

Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.     

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249–257; Björnsson et al (1996) EMBO J 15, 1696–1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA^Trp by varying the codons at positions −1 and −2 at the 5′ side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J 4, 1049–1052). The effect on readthrough by changing these −1 and −2 codons is different on the two forms of tRNA^Trp and is also dependent on the structure of EF-Tu. Readthrough by the tRNA^Trp-derived suppressor, but not wild-type tRNA^Trp, is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA^Trp are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA^Trp at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509–1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756–3760) were found not to be altered in sensitivity to the last two amino acids in the nascent peptide.