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Membrane proteins are vital for biological function and are complex to study. Even in model peptide-lipid systems, the combined influence or interaction of pairs of chemical groups still is not well understood. Disordered proteins, whether in solution or near lipid membranes, are an emerging paradigm for the initiation and control of biological function. The disorder can involve molecular orientation as well as molecular folding. This paper reports an astonishing induction of disorder when one Glu residue is introduced into a highly stable 23-residue transmembrane helix. The parent helix is anchored by a single Arg residue, tilted at a well-defined angle with respect to the DOPC bilayer normal and undergoes rapid cone precession. When Glu is introduced two residues away from Arg, with 200° (or 160°) radial separation, the helix properties change radically to exhibit a multiplicity of three or more disordered states. The helix characteristics have been monitored by deuterium (2H) NMR spectroscopy as functions of the pH and lipid bilayer composition. The disordered multistate behavior of the (Glu, Arg)-containing helix varies with the lipid bilayer thickness and pH. The results highlight a fundamental induction of protein multistate properties by a single Glu residue in a lipid membrane environment.

Transmembrane helices dominate the landscape for many membrane proteins. Often flanked by interfacial aromatic residues, these transmembrane helices also contain loops and inter-helix segments, which could help in stabilizing a transmembrane orientation. Using (2)H-NMR spectroscopy to monitor bilayer incorporated model GWALP23 family peptides, we address systematically the issue of helix fraying in relation to the dynamics and orientation of closely similar individual transmembrane helices. Adjacent to a core transmembrane helix, we inserted aromatic (Phe, Trp, Tyr, His) or non-aromatic residues (Ala, Gly) into positions 4 and 5, to examine the side-chain dependency of the transmembrane orientation, dynamics and helix integrity (extent and location of unraveling). Incorporation of (2)H-alanine labels enables one to assess the helicity of the core sequence and the peptide termini. For most of the helices, we observed substantial unwinding involving at least 3 residues at both ends. For the unique case of histidine at positions 4 and 5, an extended N-terminal unwinding was observed up to residue 7. For further investigation regarding the onset of fraying, we employed A(4,5)GWALP23 with (2)H labels at residues 4 and 5 and found that the number of terminal residues involved in the unwinding depends on bilayer thicknesses and helps to govern the helix dynamics. The combined results enable us to compare and contrast the extent of fraying for each related helix, as reflected by the deviation of experimental (2)H quadrupolar splitting magnitudes of juxta-terminal alanines A3 and A21 from those represented by an ideal helix geometry.

We have employed the peptide framework of GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide) to examine the orientation, dynamics and pH dependence of peptides having buried single or pairs of histidine residues. When residue L8 is substituted to yield GWALP23-H8, acetyl-GGALWLAH8ALALALALALWLAGA-amide, the deuterium NMR spectra of 2H-labeled core alanine residues reveal a helix that occupies a single transmembrane orientation in DLPC, or in DMPC at low pH, yet shows multiple states at higher pH or in bilayers of DOPC. Moreover, a single histidine at position 8 or 16 in the GWALP23 framework is sensitive to pH. Titration points are observed near pH 3.5 for the deprotonation of H8 in lipid bilayers of DLPC or DMPC, and for H16 in DOPC. When residues L8 and L16 both are substituted to yield GWALP23-H8,16, the 2H NMR spectra show, interestingly, no titration dependence from pH 2–8, yet bilayer thickness-dependent orientation differences. The helix with H8 and H16 is found to adopt a transmembrane orientation in thin bilayers of DLPC, a combination of transmembrane and surface orientations in DMPC, and then a complete transition to a surface bound orientation in the thicker DPoPC and DOPC lipid bilayers. In the surface orientations, alanine A7 no longer fits within the core helix. These results along with previous studies with different locations of histidine residues suggest that lipid hydrophobic thickness is a first determinant and pH a second determinant for the helical orientation, along with possible side-chain snorkeling, when the His residues are incorporated into the hydrophobic region of a lipid membrane-associated helix.

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW5 LAL8 LALALAL16 ALW19 LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific 2 H-labeled alanine residues within the central sequence for detection by solid-state 2 H NMR spectroscopy. The resulting pattern of [2 H]Ala quadrupolar splitting (Δνq ) magnitudes indicates the core helix for R8,16 GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R8,16 GWALP23 helix adopts primarily a surface orientation. The inclusion of 10-20 mol % cholesterol in DOPC bilayers drives more of the R8,16 GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.

The transmembrane helices of membrane proteins often are flanked by interfacial charged or aromatic residues that potentially help to anchor the membrane-spanning protein. For isolated single-span helices, the interfacial residues may be especially important for stabilizing particular tilted transmembrane orientations. The peptide RWALP23 (acetyl-GR(2)-AW(LA)(6)WLAR(22)A-amide) has been employed to investigate the interplay between interfacial arginines and tryptophans. Here we replace the tryptophans of RWALP23 with A5 and A19, to investigate arginines alone with respect to helix fraying and orientation in varying lipid bilayers. Deuterated alanines incorporated into the central sequence allow the orientation and stability of the core helix to be assessed by means of solid -state (2)H NMR in bilayers of DOPC, DMPC and DLPC. The helix tilt from the bilayer normal is found to increase slightly when R2 and R22 are present, and increases still further when the tryptophans W5 and W19 are replaced by alanines. The extent of helix dynamic averaging remains low in all cases. The preferred helix azimuthal rotation is essentially constant for all of the helices in each of the lipid membranes considered here. The alanines located outside of the core region of the peptide are sensitive to helical integrity. The new alanines, A5 and A19, therefore, provide new information about the length of the core helix and the onset of unraveling of the terminals. Residue A19 remains essentially on the central helix in each lipid membrane, while residues A3, A5 and A21 deviate from the core helix to an extent that depends on the membrane thickness. Differential unraveling of the two ends to expose peptide backbone groups for hydrogen bonding therefore acts together with specific interfacial side chains to stabilize a transmembrane helix.

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides. Nevertheless, the spontaneous folding process is not well understood at the molecular level. Recent findings suggest that helix fraying and loop formation may be important for overall structure, dynamics and regulation of function. Several types of membrane helices with ionizable amino acids change their topology with pH. Additionally we note that some peptides, including many that are rich in arginine, and a particular analogue of gramicidin, are able passively to translocate across cell membranes. The findings indicate that a final protein structure in a lipid-bilayer membrane is sequence-based, with lipids contributing to stability and regulation. While much progress has been made toward understanding the folding process for alpha-helical membrane proteins, it remains a work in progress. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

The Pithraj seed has been collected from Gazipur district, Bangladesh. The oil from the seed was extracted by using Soxhlet apparatus using petroleum ether extraction method. Maximum yield of oil was found to be 50 % when the process was carried out for 2.5 hours. The physicochemical properties of the extracted oil were studied. The properties of the oil reveal that the oil corresponds to diesel except acid value and sulphur content. The optimum conditions of the transesterification of the oil was 40% ethanol and 0.45% KOH at 75 0C for 1.5 hours. The optimum yield was more than 95 %.Bangladesh J. Sci. Ind. Res. 50(2), 135-142, 2015

Membrane-spanning hydrophobic alpha-helical peptides are often flanked by interfacial aromatic or charged residues that may help to stabilize the transmembrane orientation. The synthetic neutral transmembrane peptide GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide) with two interfacial Trp residues has proved to be surprisingly well-behaved with minimal dynamic averaging in a stable transmembrane orientation in lipid-bilayer membranes of varying thickness. Replacing W5 with Y5 or F5 in GWALP23 was found to yield essentially the same average tilt and dynamics in several lipid bilayers (Biochemistry, 2014, 53, 3637–3645). To investigate the tilt, dynamics and pH dependence of GWALP23 with interfacial His residues, we have substituted W5 and W19 with histidine and have incorporated 2H-Ala labels at different positions within the core helix of peptide. We have employed solid state 2H-NMR spectroscopy to evaluate the peptide tilt with respect to the bilayer normal in aligned bilayers of DMPC and DLPC at pH near 4 and 8. As the peptide exhibits well-defined 2H quadrupolar splittings from 2H-alanine methyl groups in both lipids at specific pH, we are aiming to examine the transmembrane orientations by using the Geometric Analysis of Labeled Alanines (GALA) method. Further investigations of peptide helix behavior in other lipids, and with His residues at other locations, are underway.

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that may serve as anchors to aid the stabilization of a tilted transmembrane orientation. The synthetic neutral peptide GWALP23 (acetyl-GG2ALWLALALALALALALWLAG22A-amide) with two interfacial Trp residues has proved to be surprisingly well-behaved with minimal dynamic averaging in a stable transmembrane orientation in lipid-bilayer membranes of varying thickness. To further investigate the effect of interfacial His residues, we have substituted G2 and G22 with histidine in HWALP23 (acetyl-GH2ALWLALALALALALALWLAH22A-amide). In addition, to explore the fraying or uncoiling of the ends of the peptide, we have incorporated 2H-Ala labels at positions A3 and A21 (underlined above), which are sensitive to helix integrity, outside the core region of the peptide, to compare the influence of interfacial residues on the extent of unwinding of the helix ends. Solid-state 2H NMR spectra of macroscopically aligned DOPC lipid bilayer samples and in the presence of 10% and 20% cholesterol confirmed that one or both helix ends are frayed in DOPC bilayers alone and in the presence of up to 20% cholesterol. To further understand the effects of histidine in transmembrane helices, we have also substituted W5 and W19 with histidine in GHALP23 (acetyl-GGALH5LALALALALALALH19LAGA-amide) with 2H-Ala labels at A3 and A21. “Geometric Analysis of Labeled Alanines” (GALA) shows the extent of coiling or unwinding at the terminals for HWALP23 and GHALP23. It is plausible that the helix fraying may be critical for the stability of the transmembrane helix orientation in lipid bilayer membranes.