The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry.

The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.

MscL: channeling membrane tension.

Mechanosensitive channels are integral components for the response of bacteria to osmotic shock. The mechanosensitive channel of large conductance (MscL) responds to extreme turgor pressure increase that would otherwise lyse the cellular membrane. MscL has been studied as a model mechanosensitive channel using both structural and functional approaches. We will summarize the structural data and discuss outstanding questions surrounding the gating mechanism of this homo-oligomeric channel that has ~3 nS conductance. Specifically, we will explore the following: (1) the variability in oligomeric state that has been observed, (2) the open pore size measurements, and (3) the role of the C-terminal coiled coil domain for channel function. The oligomeric state of MscL has been characterized using various techniques, with a pentamer being the predominant form; however, the presence of mixtures of oligomers in the membrane is still uncertain. In the absence of structural data for the open state of MscL, the diameter of the open state pore has been estimated by several different approaches, leading to a current estimate between 25 and 30 Å. While the C-terminal domain is highly conserved among MscL homologues, it is not required for activity in vivo or in vitro. This domain is likely to remain intact during the gating transition and perform a filtering function that retains valuable osmolytes in the cytosol. Overall, studies of MscL have provided significant insight to the field, and serve as a paradigm for the analysis of non-homologous, eukaryotic mechanosensitive channel proteins.

Structure and stability of the C-terminal helical bundle of the E. coli mechanosensitive channel of large conductance.

The crystal structure of the cytoplasmic domain (CTD) from the mechanosensitive channel of large conductance (MscL) in E. coli has been determined at 1.45 Å resolution. This domain forms a pentameric coiled coil similar to that observed in the structure of MscL from M. tuberculosis and also found in the cartilage oligomeric matrix protein (COMPcc). It contains canonical hydrophobic and atypical ionic interactions compared to previously characterized coiled coil structures. Thermodynamic analysis indicates that while the free EcMscL-CTD is less stable than other coiled coils, it is likely to remain folded in context of the full-length channel.

OCAM: a new tool for studying the oligomeric diversity of MscL channels.

We have developed a new technique to study the oligomeric state of proteins in solution. OCAM or Oligomer Characterization by Addition of Mass counts protein subunits by selectively shaving a protein mass tag added to a protein subunit via a short peptide linker. Cleavage of each mass tag reduces the total mass of the protein complex by a fixed amount. By performing limited proteolysis and separating the reaction products by size on a blue native PAGE gel, a ladder of reaction products corresponding to the number of subunits can be resolved. The pattern of bands may be used to distinguish the presence of a single homo-oligomer from a mixture of oligomeric states. We have applied OCAM to study the mechanosensitive channel of large conductance (MscL) and find that these proteins can exist in multiple oligomeric states ranging from tetramers up to possible hexamers. Our results demonstrate the existence of oligomeric forms of MscL not yet observed by X-ray crystallography or other techniques and that in some cases a single type of MscL subunit can assemble as a mixture of oligomeric states.

A reported archaeal mechanosensitive channel is a structural homolog of MarR-like transcriptional regulators.

Several archaeal mechanosensitive (MS) channels have been reported, including one from Thermoplasma volcanium designated MscTV. Here, we report the crystal structure of MscTV at 1.6-A resolution. Unexpectedly, MscTV was found to be a water-soluble protein exhibiting a winged helix-turn-helix (wHTH) motif, which is the signature of the MarR (multiple antibiotic resistance regulator) family of transcriptional regulators. A cell-based osmotic downshock functional assay demonstrated that MscTV was unable to protect a knockout strain of Escherichia coli from hypoosmotic shock, further indicating that it does not function as a MS channel. We propose this protein be renamed MLPTv for MarR-like protein from T. volcanium.

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains.

Outer membrane proteins (OMPs) of gram-negative bacteria are synthesized in the cytosol and must cross the periplasm before insertion into the outer membrane. The 17-kDa protein (Skp) is a periplasmic chaperone that assists the folding and insertion of many OMPs, including OmpA, a model OMP with a membrane embedded beta-barrel domain and a periplasmic alphabeta domain. Structurally, Skp belongs to a family of cavity-containing chaperones that bind their substrates in the cavity, protecting them from aggregation. However, some substrates, such as OmpA, exceed the capacity of the chaperone cavity, posing a mechanistic challenge. Here, we provide direct NMR evidence that, while bound to Skp, the beta-barrel domain of OmpA is maintained in an unfolded state, whereas the periplasmic domain is folded in its native conformation. Complementary cross-linking and NMR relaxation experiments show that the OmpA beta-barrel is bound deep within the Skp cavity, whereas the folded periplasmic domain protrudes outside of the cavity where it tumbles independently from the rest of the complex. This domain-based chaperoning mechanism allows the transport of beta-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.

Crystal structure of YaeT: conformational flexibility and substrate recognition.

The envelope of Gram-negative bacteria consists of inner and outer membranes surrounding the peptidoglycan wall. The outer membrane (OM) is rich in integral membrane proteins (OMPs), which have a characteristic beta barrel domain embedded in the OM. The Omp85 family of proteins, ubiquitous among Gram-negative bacteria and also present in chloroplasts and mitochondria, is required for folding and insertion of OMPs into the outer membrane. Bacterial Omp85 proteins are characterized by a periplasmic domain containing five repeats of polypeptide transport-associated (POTRA) motifs. Here we report the crystal structure of a periplasmic fragment of YaeT (the Escherichia coli Omp85) containing the first four POTRA domains in an extended conformation consistent with recent solution X-ray scattering data. Analysis of the YaeT structure reveals conformational flexibility around a hinge point between POTRA2 and 3 domains. The structure's implications for substrate binding and folding mechanisms are also discussed.

Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation.

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Here we report the crystal structure of Skp from E. coli. The structure of the Skp trimer resembles a jellyfish with alpha-helical tentacles protruding from a beta barrel body defining a central cavity. The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC, a cytosolic chaperone present in eukaria and archea, that binds unfolded substrates in its central cavity. The ability of Skp to prevent the aggregation of model substrates in vitro is independent of ATP. Skp can interact directly with membrane lipids and lipopolysaccharide (LPS). These interactions are needed for efficient Skp-assisted folding of membrane proteins. We have identified a putative LPS binding site on the outer surface of Skp and propose a model for unfolded substrate binding.

Evaluation of new linkers and synthetic methods for internal modified oligonucleotides.

Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine. The products were compared in functional assays. They performed very similarly both in a fluorescence-based melting point assay as well as in quantitative PCR. Another set of beta-actin probes were synthesized utilizing N4 [N-2-(ethylene glycol ethyl)-5-methyl]cytidine and solid-phase Tamra amidite addition at positions flanking those of the uridine. These versions gave lower T(m)s than either uridine-labeled probe and did not work as well in quantitative PCR. A control experiment using oligonucleotides with the same modified residues but without fluorophores attached revealed the same trend as the T(m) study of internal Tamra-labeled probes. Experimental details for the synthesis, purification, and testing are presented.

New reagents and methods for the synthesis of internal and 3'-labeled DNA.

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.