Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K+ channels

AbstractThe membrane location of two fragments in two different K+-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q=0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T1 and 13C–1H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. 2H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems

NMR solution structure and position of transportan in neutral phospholipid bicelles

AbstractTransportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined α-helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an α-helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts

Effect of lipid bilayer properties on the photocycle of green proteorhodopsin

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli

Dissecting the structural and chemical determinants of the "open-to-closed" motion in the mannosyltransferase PimA from Mycobacteria

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-To-β-strand and β-strand-To-α-helix transitions and (ii) an "open-To-closed"motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-To-closed"motion, with the β-phosphate group providing the high-Affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-To-closed"motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors

pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles

C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of β-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide

Lipid- and substrate-induced conformational and dynamic changes in a glycosyltransferase involved in E. coli LPS synthesis revealed by 19F and 31P NMR

WaaG is a glycosyltransferase (GT) involved in the synthesis of the bacterial cell wall, and in Escherichia coli it catalyzes the transfer of a glucose moiety from the donor substrate UDP-glucose onto the nascent lipopolysaccharide (LPS) molecule which when completed constitutes the major component of the bacterium's outermost defenses. Similar to other GTs of the GT-B fold, having two Rossman-like domains connected by a short linker, WaaG is believed to undergo complex inter-domain motions as part of its function to accommodate the nascent LPS and UDP-glucose in the catalytic site located in the cleft between the two domains. As the nascent LPS is bulky and membrane-bound, WaaG is a peripheral membrane protein, adding to the complexity of studying the enzyme in a biologically relevant environment. Using specific 5-fluoro-Trp labelling of native and inserted tryptophans and 19F NMR we herein studied the dynamic interactions of WaaG with lipids using bicelles, and with the donor substrate. Line-shape changes when bicelles are added to WaaG show that the dynamic behavior is altered when binding to the model membrane, while a chemical shift change indicates an altered environment around a tryptophan located in the C-terminal domain of WaaG upon interaction with UDP-glucose or UDP. A lipid-bound paramagnetic probe was used to confirm that the membrane interaction is mediated by a loop region located in the N-terminal domain. Furthermore, the hydrolysis of the donor substrate by WaaG was quantified by 31P NMR

Diffuse binding of Zn2+ to the denatured ensemble of Cu/Zn superoxide dismutase 1

The stability and structural properties of the metalloprotein superoxide dismutase 1 (SOD1) are found to depend critically on metal ions. Native SOD1 monomers coordinate one structural Zn2+ and one redox‐active Cu2+/1+ to the active site. To do this, the Zn2+ ions need to interact with the SOD1 protein on the denatured side of the folding barrier, prior to the formation of the folding nucleus. In this study, we have examined at residue level the nature of this early Zn2+ binding by NMR studies on the urea denatured‐state of SOD1. Nearly complete backbone chemical shift assignments were obtained in 9 M urea at physiological pH, conditions at which NMR studies are scarce. Our results demonstrate that SOD1 is predominantly unstructured under these conditions. Chemical‐shift changes upon Zn2+ titration show that denatured SOD1 retains a significant affinity to Zn2+ ions, even in 9 M urea. However, the Zn2+ interactions are not limited to the native metal‐binding ligands in the two binding sites, but are seen for all His residues. Moreover, the native Cu2+/1+ ligand H46 seems not to bind as well as the other His residues, while the nearby non‐native H43 does bind, indicating that the binding geometry is relaxed. The result suggests that the Zn2+‐binding observed to catalyze folding of SOD1 in physiological buffer is initiated by diffuse, non‐specific coordination to the coil, which subsequently funnels by ligand exchange into the native coordination geometry of the folded monomer. Altogether, this diffuse binding is a result with fundamental implications for folding of metalloproteins in general