Structure of functional A salina -- E coli hybrid ribosome by electron microscopy.

Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination -- small 30S E coli and large 60S A salina -- fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A slina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.     

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity, and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30 to 60 min of incubation provides insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high-resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.     

Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes

It is established that noncovalent complexes can be maintained both during and after electrospray and that assemblies of increasing size and complexity often lead to broadened peaks in mass spectra. This broadening arises from the tendency of large protein assemblies to form adducts with salts and is compounded when complexes are isolated directly from cells, without the full protein complement. To investigate the origins of this broadening in mass spectral peaks and to develop the optimal method for analyzing mass spectra of large protein complexes, we have carried out a systematic investigation of a series of noncovalent complexes representing a range of different sizes and architectures. We establish a positive correlation between peak width and the increased mass observed and show that this correlation is independent of the instrumental parameters employed. Using this relationship we show that we can determine masses of both 30S subunits and intact 2.3 MDa 70S ribosomes from Thermus thermophilus. The masses of both particles are consistent with multiple populations of ribosomes. To identify these various populations we combine simulated mass spectra of ribosomes, with and without the full protein complement, and estimate the extent of adducts from our study of known complexes. The results allow us to determine the contribution of the different subpopulations to the overall mass spectrum. We confirm the existence of these subpopulations using tandem mass spectrometry of intact 30S subunits. Overall, the results show that, rather than uniform particles, gas-phase ribosomes consist of a number of discrete populations. More generally, the results establish a rigorous procedure for accurate mass measurement and spectral analysis of heterogeneous macromolecular assemblies.     

Capillary electrophoresis of affinity complexes between subviral 80S particles of human rhinovirus and monoclonal antibody 2G2

Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in vitro, upon exposure to a temperature between 50 and 56 degrees C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH 8.3, 100 mM) containing 10 mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb 2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb 2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype 2 into 80S particles upon incubation at temperatures between 40 and 65 degrees C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb 2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody.     

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m2G966 and m?C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low μM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.     

Separation of ribosomal subunits on Trisacryl GF 2000

The separation of rat liver and E. coli ribosomal subunits was attempted on Trisacryl GF 2000. Contrary to experiments with Sepharose 4B and Bio-Gel A-15 the 60S mammalian subunit did not bind to the resin at 4 degrees C but eluted within the column volume ahead of the 40S subunit. Puromycin, however, used to prepare the subunits, which on the agarose gels had eluted at the total column volume, exhibited anomalous retardation on the Trisacryl resin. Trisacryl therefore behaves as the more non-polar resin, and the binding of 60S subunits to agarose gels is a result of hydrophilic interaction.     

Redistribution of intramembrane particles of human erythrocytes induced by HVJ (Sendai virus): a prerequisite for the virus-induced cell fusion.

Aggregation of intramembrane particles of human erythrocytes was found to be induced by HVJ (Sendai virus) under conditions which lead to cell fusion. Degree of polyerythrocyte formation was compared under a variety of conditions with extent of cluster formation observed with the same preparations. Both structural changes of the membranes, ie, fusion and clustering of the particles, behaved very similarly under widely different virus-to-cell ratios and over the time course of cell fusion. Furthermore, by inclusion of high concentrations of antispectrin antibodies within the ghosts, inhibition of clustering of intramembrane particles and hindrance of virus-induced cell fusion were found to occur simultaneously. Antibodies by themselves did not induce aggregation of particles under isotonic conditions, whereas particle clustering could be induced under hypotonic conditions at antibody concentrations causing partial cross-linking of spectrin molecules. In conclusion, clustering of intramembrane particles seems to be required for virus-induced fusion of human erythrocytes.     

RNA PROTEIN CROSSLINKS INTRODUCED INTO E. coli RIBOSOMES BY USE OF THE INTRINSIC PROBE 4-THIOURIDINE

Abstract— 70S Ribosome substituted by the uridine photoactivable analogue 4-thiouridine has been prepared by an in vivo method (substitution level 4.5%). The r-proteins crosslinked to 16S and 23S rRNA before and after 366-nm photoactivation were identified. Proteins S2-S7-S9/11-S18 are found linked to 16S RNA in dark-prepared 30S subunits. Illumination increases uniformly their binding by a factor of 2.5. Similarly, proteins L5-L15-L18-L23-L28-L32 are found crosslinked to 23S RNA in dark-prepared 50S subunits. Photoactivation increases their binding but in addition promotes the covalent linking of proteins L1-L3-L4.     

Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry

Direct analysis of intact proteins on a chromatographic time scale is demonstrated on a modified linear ion trap mass spectrometer using sequential ion/ion reactions, electron transfer and proton transfer, to dissociate the sample and to convert the resulting peptide fragments to a mixture of singly and doubly charged species. Proteins are converted to gas-phase, multiply-charged, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with even-electron benzoate anions. M/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 amino acids at both the N-terminus and the C-terminus of the protein. This information, along with the measured mass of the intact protein, are employed to identify known proteins and to detect the presence of post-translational modifications. In this study, we analyze intact proteins from the Escherchia coli 70S ribosomal protein complex and identify 46 of the 55 known unique components in a single, 90 min, on-line, chromatography experiment. Truncated versions of the above proteins along with several post-translational modifications are also detected.     

Structural studies of E. coli ribosomes by spectroscopic techniques: a specialized review

We present a review on our interdisciplinary line of research based on strategies of molecular biology and biophysics. These have been applied to the study of the prokaryotic ribosome of the bacterium Escherichia coli. Our investigations on this organelle have continued for more than a decade and we have adopted different spectroscopic biophysical techniques such as: dielectric and fluorescence spectroscopy as well as light scattering (photon correlation spectroscopy). Here we report studies on the whole 70S ribosomes and on the separated subunits 30S and 50S. Our results evidence intrinsic structural features of the subunits: the small shows a more "floppy" structure, while the large one appears to be more rigid. Also, an inner "kernel" formed by the RNA/protein association is found within the ribosome. This kernel is surrounded by a ribonucleoprotein complex more exposed to the solvent. Initial analyses were done on the so called Kaldtschmit-Wittmann ribosome: more recently we have extended the studies to the "tight couple" ribosome known for its better functional performance in vitro. Data evidence a phenomenological correlation between the differential biological activity and the intrinsic structural properties of the two-ribosome species. Finally, investigations were also conducted on particles treated at sub-denaturing temperatures and on ribosomes partially deproteinized by salt treatment (ribosomal cores). Results suggest that the thermal treatment and the selective removal of proteins cause analogous structural alterations.     

Chromatographic method for the isolation of active 40S and 60S subunits from rat liver polyribosomes

A rapid and simple procedure for isolation of 40S and 60S ribosomal subunits by ion-exchange column chromatography is described. The dissociated ribosomes can be separated and non-ribosomal proteins and low-molecular-weight substances removed. An assessment by physicochemical and functional criteria showed that the ribosomal subunits obtained are active and sufficiently homogeneous.     

Localization of noncovalent complexes in MALDI-preparations by CLSM

The unambiguous detection of noncovalent complexes (NCCs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still a far cry from being routine. For protein NCCs such as their quaternary structure it has been reported that signals of the intact complex are only obtained for the first or at most the first few laser exposures of a given sample area. This observation was called the first-shot phenomenon. In the present study, this first-shot phenomenon has been investigated for the hexameric protein complex allophycocyanine (APC) by two independent methods, MALDI-MS with a (nearly) pH-neutral matrix 6-aza-2-thiothymine (6-ATT) and by imaging the fluorescence of the complex in APC-6-ATT preparations by confocal laser scan microscopy (CLSM). The intact APC heterohexamer loses its visible fluorescence upon dissociation into its subunits. Both methods consistently show that intact APC complexes are precipitated at the matrix crystal surface, but dissociate upon incorporation into the matrix crystals.     

PROTEIN-RNA CROSSLINKS IN RIBOSOMES: ULTRAVIOLET ACTION SPECTRA. COMPARISON WITH INACTIVATION ACTION SPECTRUM

Abstract— RNA-protein crosslinking by UV of different wavelengths was studied in 70S E. coli ribosomes by three techniques: sucrose gradient centrifugation in the presence of sodium dodecyl sulfate (SDS), RNA solubilization in LiCI-urea concentrated solutions and RNA adsorption on nitrocellulose filters in the presence of SDS.
The centrifugational technique shows that the crosslinking reaction occurs in two steps, the first one corresponding to the fixation of a few protein molecules on 16 or 23 s RNAs and the second one corresponding to extensive RNA-protein crosslinking so that most protein molecules are no longer released by SDS from 30S and 50S subunits.
The initial rates for the first step of crosslinking were evaluated by the solubilization and adsorption techniques at 7 (or 6) wavelengths of irradiation between 223 and 290 mm. The action spectrum for RNA solubilization in LiCl-urea is perturbed at 223 nm by the breakage of protein chains. The action spectrum for retention on nitrocellulose filters seems to be exempt of this defect. It corresponds at high wavelengths to a nucleic chromophore and at low wavelengths to a proteic one. This means that RNA-protein crosslinking may occur through RNA and protein excitation. The similarity between the action spectrum for RNA retention on nitrocellulose filters and the action spectrum for inactivation of ribosomal synthesis activity suggests that RNA-protein crosslinking may be responsible for inactivation of ribosomes by UV.     

Isoelectric focusing studies of concanavalin A and the lentil lectin

Isoelectric focusing (IEF) of metallized and demetallized preparations of concanavalin A (Con A) consisting of either intact or fragmented subunits shows different band patterns. Metallized Con A consisting of intact polypeptide chains (intact Con A) has an isoelectric point (pI) 8.35. Metallized preparations consisting of fragmented chains (fragmented Con A) show three bands with pI values 8.0, 7.8 and 7.7. Demetallized intact Con A (intact apoCon A) has a pI of 6.5, however, it undergoes pH dependent association during IEF under certain conditions, which gives rise to multiple bands. Ampholyte-mediated demetallization of intact and fragmented Con A and subsequent aggregation of the apoprotein results in multiple bands during IEF in the presence of the pH range 3 to 10 ampholytes. However, ampholytes of the pH range 7 to 9 do not demetallize the proteins and show a single band with intact Con A. The pI of intact Con A remains essentially the same in the presence of inhibitory sugar. Furthermore, different moleculars forms of Con A, including locked and unlocked conformers of intact apoCon A, and the dimeric and tetramic states of both intact Con A and intact apoCon A have been identified and their pI values determined. IEF of the lentil isoelectins, LcH-A and LcH-B, shows single bands of pI 8.5 and 9.0, respectively. However, the native lectin mixture gives rise to an additional band of pI 8.8 due to a hybrid protein formed by ampholyte-mediated subunit exchange between the isolectins.     

Electrophoretic analysis of phosphorylation of the yeast 20S proteasome

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.     

Ribosome Subunit Stapling for Orthogonal Translation in E. coli

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl‐transferase center, for unnatural polymer synthesis in cells.     

Three-dimensional electron cryomicroscopy of ribosomes

Single particle electron cryomicroscopy is nowadays routinely used to generate three-dimensional structural information of ribosomal complexes without the need of crystallization. A large number of structures of functional important ribosomal complexes have thus been determined using this technique. In E. coli 70S ribosomes all three tRNA binding sites could be localized. The ternary complex of EF-TutRNAGTP that delivers the tRNA to the ribosome was directly visualized in a ribosomal complex blocked by the antibiotic kirromycin. Three different functional states of translocation have been studied and the respective EF-G binding sites have been mapped. The level of resolution achievable with electron cryomicroscopy allows conformational changes in the domain structures of elongation factors to be modelled in terms of rigid body movements. Structural information on eukaryotic ribosomes is also available for yeast and mammalian 80S ribosomes. The structural differences between rabbit 80S and E. coli 70S ribosomes could be interpreted in terms of ribosomal RNA expansion segments in the 18S and 23S RNA. The EF-G homologue EF2 was mapped analysing the structure of an 80SEF2sodarin complex and most recently the binding of a hepatitis C virus IRES element to a yeast 40S subunit has been studied. The first electron cryomicroscopical 3D reconstructions have further been used to overcome the initial phasing problems in X-ray crystallographic studies of the ribosome facilitating structure determination of the recent atomic resolution structures of the 30S and 50S ribosomal subunits. In turn, the knowledge of the atomic structure of the ribosome makes detailed interpretations of cryo-EM maps possible at approximately 20 A resolution.     

Identification of Proteins Using Affinity Affi-Gel Simultaneously with Acrylamide Electrophoresis

Abstract

This work presents a method in which a desired protein may be separated from a biological protein preparation and identified by highly specific monoclonal antibody. The desired protein may be isolated from crude protein preparations by this method. The protein remains intact and stable throughout the procedure. This method utilizes common nonreducing conditions of polyacrylamide gel electrophoresis (PAGE), and Western Blot transfer of protein. This isolation of the desired protein can be accomplished for a microgram or tens of micrograms of material. The application of Western blot technique with monoclonal antibody permits the highest specificity in terms of identifying the target protein. In addition to identifying the target protein the method can isolate sufficient amounts for further characterization.