Noncovalent Host-Guest Complexes of Artemisinin with α-, ß-, and γ- Cyclodextrin Examined by Structural Mass Spectrometry Strategies.

Cyclodextrins (CDs) are a family of macrocyclic oligosaccharides with amphiphilic properties, which can improve the stability, solubility, and bioavailability of therapeutic compounds. There has been growing interest in the advancement of efficient and reliable analytical methods that assist with elucidating CD host-guest drug complexation. In this study, we investigate the noncovalent ion complexes formed between naturally occurring dextrins (αCD, ßCD, γCD, and maltohexaose) with the poorly water-soluble antimalarial drug, artemisinin, using a combination of ion mobility-mass spectrometry (IM-MS), tandem MS/MS, and theoretical modeling approaches. This study aims to determine if the drug can complex within the core dextrin cavity forming an inclusion complex or nonspecifically bind to the periphery of the dextrins. We explore the use of group I alkali earth metal additives to promote the formation of various noncovalent gas-phase ion complexes with different drug/dextrin stoichiometries (1:1, 1:2, 1:3, 1:4, and 2:1). Broad IM-MS collision cross section (CCS) mapping (n > 300) and power-law regression analysis were used to confirm the stoichiometric assignments. The 1:1 drug:αCD and drug:ßCD complexes exhibited strong preferences for Li+ and Na+ charge carriers, whereas drug:γCD complexes preferred forming adducts with the larger alkali metals, K+, Rb+, and Cs+. Although the ion-measured CCS increased with cation size for the unbound artemisinin and CDs, the 1:1 drug:dextrin complexes exhibit near-identical CCS values regardless of the cation, suggesting these are inclusion complexes. Tandem MS/MS survival yield curves of the [artemisinin:ßCD + X]+ ion (X = H, Li, Na, K) showed a decreased stability of the ion complex with increasing cation size. Empirical CCS measurements of the [artemisinin:ßCD + Li]+ ion correlated with predicted CCS values from the low-energy theoretical structures of the drug incorporated within the ßCD cavity, providing further evidence that gas-phase inclusion complexes are formed in these experiments. Taken together, this work demonstrates the utility of combining analytical information from IM-MS, MS/MS, and computational approaches in interpreting the presence of gas-phase inclusion phenomena.

Outer membrane ß-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA.

Outer membrane ß-barrel proteins (OMPs) are crucial for numerous cellular processes in prokaryotes and eukaryotes. Despite extensive studies on OMP biogenesis, it is unclear why OMPs require assembly machineries to fold into their native outer membranes, as they are capable of folding quickly and efficiently through an intrinsic folding pathway in vitro. By investigating the folding of several bacterial OMPs using membranes with naturally occurring Escherichia coli lipids, we show that phosphoethanolamine and phosphoglycerol head groups impose a kinetic barrier to OMP folding. The kinetic retardation of OMP folding places a strong negative pressure against spontaneous incorporation of OMPs into inner bacterial membranes, which would dissipate the proton motive force and undoubtedly kill bacteria. We further show that prefolded ß-barrel assembly machinery subunit A (BamA), the evolutionarily conserved, central subunit of the BAM complex, accelerates OMP folding by lowering the kinetic barrier imposed by phosphoethanolamine head groups. Our results suggest that OMP assembly machineries are required in vivo to enable physical control over the spontaneously occurring OMP folding reaction in the periplasm. Mechanistic studies further allowed us to derive a model for BamA function, which explains how OMP assembly can be conserved between prokaryotes and eukaryotes.

Outer Membrane Protein Folding and Topology from a Computational Transfer Free Energy Scale.

Knowledge of the transfer free energy of amino acids from aqueous solution to a lipid bilayer is essential for understanding membrane protein folding and for predicting membrane protein structure. Here we report a computational approach that can calculate the folding free energy of the transmembrane region of outer membrane ß-barrel proteins (OMPs) by combining an empirical energy function with a reduced discrete state space model. We quantitatively analyzed the transfer free energies of 20 amino acid residues at the center of the lipid bilayer of OmpLA. Our results are in excellent agreement with the experimentally derived hydrophobicity scales. We further exhaustively calculated the transfer free energies of 20 amino acids at all positions in the TM region of OmpLA. We found that the asymmetry of the Gram-negative bacterial outer membrane as well as the TM residues of an OMP determine its functional fold in vivo. Our results suggest that the folding process of an OMP is driven by the lipid-facing residues in its hydrophobic core, and its NC-IN topology is determined by the differential stabilities of OMPs in the asymmetrical outer membrane. The folding free energy is further reduced by lipid A and assisted by general depth-dependent cooperativities that exist between polar and ionizable residues. Moreover, context-dependency of transfer free energies at specific positions in OmpLA predict regions important for protein function as well as structural anomalies. Our computational approach is fast, efficient and applicable to any OMP.

Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm.

Thermodynamic stabilities are pivotal for understanding structure-function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure-stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.

Structural elements of the mitochondrial preprotein-conducting channel Tom40 dissolved by bioinformatics and mass spectrometry.

Most mitochondrial proteins are imported into mitochondria from the cytosolic compartment. Proteins destined for the outer or inner membrane, the inter-membrane space, or the matrix are recognized and translocated by the TOM machinery containing the specialized protein import channel Tom40. The latter is a protein with ß-barrel shape, which is suggested to have evolved from a porin-type protein. To obtain structural insights in the absence of a crystal structure the membrane topology of Tom40 from Neurospora crassa was determined by limited proteolysis combined with mass spectrometry. The results were interpreted on the basis of a structural model that has been generated for NcTom40 by using the structure of mouse VDAC-1 as a template and amino acid sequence information of approximately 270 different Tom40 and approximately 480 VDAC amino acid sequences for refinement. The model largely explains the observed accessible cleavage sites and serves as a structural basis for the investigation of physicochemical properties of the ensemble of our Tom40 sequence data set. By this means we discovered two conserved polar slides in the pore interior. One is possibly involved in the positioning of a pore-inserted helix; the other one might be important for mitochondrial pre-sequence peptide binding as it is only present in Tom40 but not in VDAC proteins. The outer surface of the Tom40 barrel reveals two conserved amino acid clusters. They may be involved in binding other components of the TOM complex or bridging components of the TIM machinery of the mitochondrial inner membrane.

Functional refolding and characterization of two Tom40 isoforms from human mitochondria.

Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

The Role of a Destabilized Membrane for OMP Insertion.

Here we describe the procedures used in our laboratory for the in vitro investigation of the apparent folding kinetics as well as the folding efficiencies of outer membrane proteins (OMPs). Because microbial OMPs display a change in their gel migration upon folding, the usage of traditional gel electrophoresis is a standard method of folding analysis. Additional aspects of the method we detail herein include the preparation and storage of OMP stocks, the setup procedures for a folding reaction, and the analysis of fraction folded from scanned gel images.

Improving the resistance of a eukaryotic ß-barrel protein to thermal and chemical perturbations.

ß-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) ß-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the "mitochondrial porin family" and main constituent of the mitochondrial protein-conducting channel TOM (translocase of the outer membrane). Using a physical interaction model, TmSIP, for ß-barrel membrane proteins, we have identified three unfavorable ß-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable ß-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of ß-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.