On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID.

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.

Chaperonin-catalyzed rescue of kinetically trapped states in protein folding.

GroEL and GroES form a chaperonin nano-cage for single protein molecules to fold in isolation. The folding properties that render a protein chaperonin dependent are not yet understood. Here, we address this question using a double mutant of the maltose-binding protein DM-MBP as a substrate. Upon spontaneous refolding, DM-MBP populates a kinetically trapped intermediate that is collapsed but structurally disordered. Introducing two long-range disulfide bonds into DM-MBP reduces the entropic folding barrier of this intermediate and strongly accelerates native state formation. Strikingly, steric confinement of the protein in the chaperonin cage mimics the kinetic effect of constraining disulfides on folding, in a manner mediated by negative charge clusters in the cage wall. These findings suggest that chaperonin dependence correlates with the tendency of proteins to populate entropically stabilized folding intermediates. The capacity to rescue proteins from such folding traps may explain the uniquely essential role of chaperonin cages within the cellular chaperone network.

Structural features of fusogenic model transmembrane domains that differentially regulate inner and outer leaflet mixing in membrane fusion.

The transmembrane domains of fusion proteins are known to be important for their fusogenic activity. In an effort to systematically investigate the structure/function relationships of transmembrane domains we had previously designed LV-peptides that mimic natural fusion protein TMDs in their ability to drive fusion after incorporation into liposomal membranes. Here, we investigate the impact of different structural features of LV-peptide TMDs on inner and outer leaflet mixing. We find that fusion driven by the helical peptides involves a hemifusion intermediate as previously seen for natural fusion proteins. Helix backbone dynamics enhances fusion by selectively promoting outer leaflet mixing. Furthermore, the hydrophobic length of the peptides as well as covalent attachment of long acyl chains affects outer and inner leaflet mixing to different extents. Different structural features of transmembrane domains thus appear to differentially influence the rearrangements of lipids in fusion initiation and the hemifusion-to-fusion transition. The relevance of these findings in respect to the function of natural fusion proteins is discussed.

Stabilization of conformationally dynamic helices by covalently attached acyl chains.

Acylation of proteins is known to mediate membrane attachment and to influence subcellular sorting. Here, we report that acylation can stabilize secondary structure. Circular dichroism spectroscopy showed that N-terminal attachment of acyl chains decreases the ability of an intrinsically flexible hydrophobic model peptide to refold from an alpha-helical state to beta-sheet in response to changing solvent conditions. Acylation also stabilized the membrane-embedded alpha-helix. This increase of global helix stability did not result from decreased local conformational dynamics of the helix backbone as assessed by deuterium/hydrogen-exchange experiments. We concluded that acylation can stabilize the structure of intrinsically dynamic helices and may thus prevent misfolding.

Sequence-specific conformational dynamics of model transmembrane domains determines their membrane fusogenic function.

The transmembrane domains of fusion proteins are known to be functionally important and display an overabundance of helix-destabilizing Ile and Val residues. In an effort to systematically study the relationship of fusogenicity and helix stability, we previously designed LV peptides, a low-complexity model system whose hydrophobic core consists of Leu and Val residues at different ratios. The ability of LV peptides to fuse membranes increases with the content of helix-destabilizing residues. Here, we monitored the kinetics of amide deuterium/hydrogen exchange of LV-peptide helices to probe their conformational dynamics. The kinetics indeed increases strongly with the content of helix-destabilizing residues and is likely to reflect local fluctuations of the helix backbones as all peptides exhibit uncorrelated exchange and contain subpopulations of amide deuterium atoms that exchange with different velocities. Interestingly, helices whose amide deuterium atoms are shifted from slower to faster subpopulations are more fusogenic. Novel peptide variants in which Val residues are concentrated at peripheral or central domains of the hydrophobic core were designed to map functionally relevant helix subdomains. Their structural and functional analysis suggests that dynamic domains close to the helix termini are more relevant for fusogenicity than central domains but cooperate with the latter to achieve strong fusogenicity.

Sequence-specific conformational flexibility of SNARE transmembrane helices probed by hydrogen/deuterium exchange.

SNARE proteins mediate fusion of intracellular eukaryotic membranes and their alpha-helical transmembrane domains are known to contribute to lipid bilayer mixing. Synthetic transmembrane domain peptides were previously shown to mimic the function of SNARE proteins in that they trigger liposome fusion in a sequence-specific fashion. Here, we performed a detailed investigation of the conformational dynamics of the transmembrane helices of the presynaptic SNAREs synaptobrevin II and syntaxin 1a. To this end, we recorded deuterium/hydrogen-exchange kinetics in isotropic solution as well as in the membrane-embedded state. In solution, the exchange kinetics of each peptide can be described by three different classes of amide deuteriums that exchange with different rate constants. These are likely to originate from exchange at different domains of the helices. Interestingly, the rate constants of each class vary with the TMD sequence. Thus, the exchange rate is position-specific and sequence-specific. Further, the rate constants correlate with the previously determined membrane fusogenicities. In membranes, exchange is retarded and a significant proportion of amide hydrogens are protected from exchange. We conclude that the conformational dynamics of SNARE TMD helices is mechanistically linked to their ability to drive lipid mixing.

pH-Activated fusogenic transmembrane LV-peptides.

LV-peptides mimic the in vitro fusogenicity of synthetic fusion protein transmembrane domains. The original versions of these peptides consist of a variable hydrophobic core (containing leucine and/or valine residues (LV)) that is flanked by invariant lysine triplets at both termini. Previously, peptide fusogenicity was correlated with the structural plasticity of their hydrophobic cores. Here, we examined the functional importance of positively charged flanking residues. To this end, we determined the fusogenicities of peptide variants that contain terminal His and/or Lys triplets. Interestingly, liposome fusion by peptides with His triplets was triggered by acidic pH. The pH dependence of fusion is reflected by a sigmoidal titration curve whose midpoint is close to the pKa value of histidine. Thus, only peptides with positively charged residues at both termini are fusogenic. The previously established dependence of fusogenicity on the sequence of the hydrophobic peptide core of Lys-flanked LV-peptides was preserved with the His-flanked versions at low pH. We propose that the structural flexibility of the core region as well as positive terminal charges are required for LV-peptide function in lipid mixing. In a potential practical application, the pH-dependent LV-peptides might prove to be useful in the lipofection of eukaryotic cells.

Self-interaction of a SNARE transmembrane domain promotes the hemifusion-to-fusion transition.

SNARE proteins mediate intracellular fusion of eukaryotic membranes. Some SNAREs have previously been shown to dimerise via interaction of their transmembrane domains. However, the functional significance of these interactions had remained unclear. Here, we show that mutating alternate faces of the transmembrane helix of the yeast vacuolar Q-SNARE Vam3p reduces the ability of the full-length protein to induce contents mixing in yeast vacuole fusion to different extents. Examination of liposome fusion induced by synthetic transmembrane domains revealed that inner leaflet mixing is delayed relative to outer leaflet mixing, suggesting that fusion transits through a hemifusion intermediate. Interestingly, one of the mutations impaired inner leaflet mixing in the liposome system. This suggests that the defect seen in vacuolar contents mixing is due to partial arrest of the reaction at hemifusion. Since covalent dimerisation of this mutant recovered wild-type behaviour, homodimerisation of a SNARE transmembrane domain appears to control the transition of a hemifusion intermediate to complete lipid mixing.