Using nanoelectrospray ion mobility spectrometry (GEMMA) to determine the size and relative molecular mass of proteins and protein assemblies: a comparison with MALLS and QELS.

The determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool.

Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function.

SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs. We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controlling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its alpha-helical content. The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2-NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation.

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.

QSR1, an essential yeast gene with a genetic relationship to a subunit of the mitochondrial cytochrome bc1 complex, codes for a 60 S ribosomal subunit protein.

QSR1 (quinol-cytochrome c reductase subunit-requiring) is a highly conserved, essential gene in Saccharomyces cerevisiae that was identified through a synthetic lethal screen by its genetic relationship to QCR6, the gene for subunit 6 (Qcr6p) of the mitochondrial cytochrome bc1 complex. The function of the QSR1-encoded protein (Qsr1p) and its relationship to the QCR6-encoded protein are unknown. When yeast cell lysates are fractionated by density gradient centrifugation, Qsr1p separates from organelles and sediments with a uniformly sized population of particles that are similar to eukaryotic ribosomes upon velocity gradient centrifugation. When 40 S and 60 S ribosomal subunits are separated on velocity gradients, Qsr1p is found exclusively with the 60 S subunits, where it is a stoichiometric component. Extracts prepared from qsr1-1 cells are defective in in vitro translation assays relative to the wild type. In yeast cell lysates in which QCR6 rescues an otherwise lethal qsr1-1 mutation, Qcr6p is found only in mitochondria, both in respiratory-competent cells and in rho0 cells in which the bc1 complex is no longer present. These results suggest that suppression of the qsr1-1 mutation by QCR6 occurs by a trans-relationship across the outer mitochondrial membrane.

Diversity among abortion strains of Chlamydia psittaci demonstrated by inclusion morphology, polypeptide profiles and monoclonal antibodies.

Twenty eight C. psittaci abortion strains had been previously classified in to 4 immunologically distinct groups on the basis of cross-protection experiments in a mouse model. To identify the molecular basis of their immunological divergence 4 representative strains were investigated by cellular, molecular and immunological techniques. An identical pattern was obtained by Alul digestion of the amplified major outer membrane protein gene (MOMP) by the polymerase chain reaction (PCR) of the 4 strains. However, inclusion morphology and polypeptide profiles clearly distinguished one strain, named LLG, and its homologous strain POS from the other prototypes by the presence of a unique protein at 26.5 kDa and the absence of a polypeptide at 23 kDa. Six out of 10 monoclonal antibodies (mAbs) raised against abortion strains failed to react with inclusions of the 2 strains. All 6 mAbs reacted with the chlamydial outer membrane complex (COMC). Two of these mAbs, one against the MOMP and one against an antigen at 90 kDa, did not react with immunoblots of LLG and POS. The data provide direct demonstration of the existence of strain variation in the field and classify strains LLG and POS as a distinct C. psittaci serotype 1-subtype. The antigenic diversity among abortion strains should be taken into consideration when designing a subunit vaccine.

Heat shock proteins in the moderately halophilic bacterium Deleya halophila: protective effect of high salt concentration against thermal shock.

The moderately halophilic eubacterium Deleya halophila grown in medium containing 1 M or 2.5 M NaCl was heat-shocked at various temperatures and the electrophoretic patterns of pulse-labelled proteins were examined. Several polypeptides were induced (heat shock proteins, or hsp) at all temperatures. However, the level of induction of some hsp was dependent on the severity of the thermal shock as well as the salt concentration of the growth medium. Time course studies revealed that synthesis of some of the hsp was transient when cells were grown in 1 M NaCl, while growth at 2.5 M NaCl resulted in the synthesis of most of the hsp at almost maximal level for at least 60 min following temperature shift-up. When cells were returned to normal growth temperature (30 degrees C) after a heat shock treatment (47 degrees C for 5 min), normal protein synthesis resumed faster when cells were grown in 1 M than in 2.5 M NaCl. During the recovery period, several major hsp appeared to be synthesized at near maximal level at both salt concentrations.