Protein folding observed by time-resolved synchrotron x-ray scattering. A feasibility study.

A solution to the "protein folding" problem, the successful prediction of tertiary and quaternary protein structure from amino acid or gene sequence, would be a major advance in biology and biotechnology. Knowledge of any intermediate structure between fully unwound and folded would aid folding calculations. The use of high intensity synchrotron x-rays from the SUNY X21 beamline at National Synchrotron Light Source has been investigated as a probe of structural changes during protein folding and unfolding in solution. A temperature jump apparatus was used to study thermally-induced folding and unfolding. Scattering of solutions of myoglobin in the angular range 20 = 1-50 mrad. was measured during temperature jumps between 26 and 76 degrees C. There are clear signs of time/temperature-dependent structural changes, in the small angle region, consistent with those from other equilibrium techniques. Analysis indicates that this experimental technique can be extended to the higher angle region where theoretical calculations indicate more detailed structural information, for example when alpha-helix formation, is present.     

Detection of functional ligand-binding events using synchrotron x-ray scattering

Small-molecule ligands that change the structure of a protein are likely to affect its function, whereas those causing no structural change are less likely to be functional. Wide-angle x-ray scattering (WAXS) can be easily carried out on proteins and small molecules in solution in the absence of chemical tags or derivatives. The authors demonstrate that WAXS is a sensitive probe of ligand binding to proteins in solution and can distinguish between nonfunctional and productive binding. Furthermore, similar ligand-binding modes translate into similar scattering patterns. This approach has high potential as a novel, generic, low-throughput assay for functional ligand binding.     

Preferential inhibition of phosphatidyl ethanolamine synthesis in E. coli by alcohols.

Growth of E. coli in the presence of alcohols of chain lengths 1 through 8 results in an increase in the relative abundance of phosphatidyl glycerol. This results primarily from the preferential inhibition of phosphatidyl ethanolamine synthesis. This inhibition appears to be unrelated to membrane fluidity or to changes in fatty acid composition caused by alcohols. Alcohol-induced changes in total fatty acid composition are reflected in all phospholipid classes. Phosphatidyl serine synthetase is proposed as the most likely site for the effects of alcohols on phospholipid synthesis.     

Comparative structural studies of Vpu peptides in phospholipid monolayers by x-ray scattering

Vpu is an 81-residue HIV-1 accessory protein, its transmembrane and cytoplasmic domains each responsible for one of its two functions. Langmuir monolayers of phospholipid incorporating a membrane protein with a unidirectional vectorial orientation, on a semiinfinite aqueous subphase, provide one "membranelike" environment for the protein. The cytoplasmic domain's interaction with the surface of the phospholipid monolayer in determining the tertiary structure of the peptide within the monolayer was investigated, employing a comparative structural study of Vpu with its submolecular fragments Tm and TmCy truncated to different extents in the cytoplasmic domain, via synchrotron x-ray scattering utilizing a new method of analysis. Localizations of the transmembrane and cytoplasmic domains within the monolayer profile structure were similar for all three proteins, the hydrophobic transmembrane helix within the hydrocarbon chain region tilted with respect to the monolayer plane and the helices of the cytoplasmic domains lying on the surface of the headgroups parallel to the monolayer plane. The thickness of the hydrocarbon chain region, determined by the tilt of the hydrocarbon chains and transmembrane domain with respect to the monolayer plane, was slightly different for Tm, TmCy, and Vpu systematically with protein/lipid mole ratio. Localization of the helices in the cytoplasmic domains of the three proteins relative to the headgroups depends on their extents and amphipathicities. Thus, the interaction of the cytoplasmic domain of Vpu on the surface may affect the tilt of the transmembrane helix within the hydrocarbon chain region in determining its tertiary structure in the membrane.     

Conformation of peptides in lipid membranes studied by x-ray grazing incidence scattering

Although the antimicrobial, fungal peptide alamethicin has been extensively studied, the conformation of the peptide and the interaction with lipid bilayers as well as the mechanism of channel gating are still not completely clear. As opposed to studies of the crystalline state, the polypeptide structures in the environment of fluid bilayers are difficult to probe. We have investigated the conformation of alamethicin in highly aligned stacks of model lipid membranes by synchrotron-based x-ray scattering. The (wide-angle) scattering distribution has been measured by reciprocal space mappings. A pronounced scattering signal is observed in samples of high molar peptide/lipid ratio which is distinctly different from the scattering distribution of an ideal helix in the transmembrane state. Beyond simple models of ideal helices, the data is analyzed in terms of models based on atomic coordinates from the Brookhaven Protein Data Bank, as well as from published molecular dynamics simulations. The results can be explained by assuming a wide distribution of helix tilt angles with respect to the membrane normal and a partial insertion of the N-terminus into the membrane.     

Structural characterization of lactate dehydrogenase dissociation under high pressure studied by synchrotron high-pressure small-angle X-ray scattering.

The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray scattering (SAXS). The SAXS results are interpreted in terms of the dissociation and association of LDH within a compression and decompression cycle and its temperature dependence. LDH consists of four identical subunits. At 120 MPa and 25 degrees C, 50% of the LDH dissociates into subunits, while at 10 degrees C, this occurs at 78 MPa. The hysteresis in the dissociation and association under pressure was confirmed in terms of the radius of gyration and was seen to be more conspicuous at low temperature. Forward scattering, I(0)/C, which is proportional to molecular weight, showed that LDH dissociated into dimer (not monomer) subunits under pressure. The application of high pressure to dissociated dimers induced irreversible aggregation. This result is in sharp contrast with the result of fluorescence spectroscopy suggesting a dissociated monomer [King, L., and Weber, G. (1986) Biochemistry 25, 3637-3640]. As for structural change after reassociation, there was little structural difference between native and drifted LDH. The difference was smaller than the structure change by ligand binding. At 200 MPa, the presence of five scattering peaks in the medium-angle region indicates that the dissociated dimer does not have a molten globule-like structure but a core structure. We propose a model of the dissociated dimer, based on the SAXS profile, in which the volume is reduced without disrupting the core structure.     

Synthesis of an unsaturated diether analogue of phosphatidyl ethanolamine

A synthesis of racemic diether analogues of glyceryl phosphatides is reported which is applicable to unsaturated aliphatic substituents. The "oleyl" diether analogue of phosphatidyl ethanolamine has been synthesized.     

Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of arbutin on the dipole potential and area per lipid of ester and ether phosphatidylcholine and phosphatidyl ethanolamine monolayers

The present results report for the first time a systematic study of the effect of arbutin on the dipole potential of lipid membranes. The dipole potential and the area per lipid were measured in monolayers of dimyristoylphosphatidylcholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoylphosphatidylethanolamine (DMPE) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE), spread on aqueous solutions of different concentrations of arbutin. The decrease of the dipole potential of DMPC, both in condensed and expanded monolayers, is parallel to an increase in the area per lipid. In contrast, for dietherPC, the area per lipid is not affected, in spite of the fact that arbutin is also able to decrease the dipole potential in a less drastic extent. In the case of DMPE, the response is similar to that observed with dietherPC: the dipole potential decreases, while the area per lipid remains unchanged. However, when the carbonyl groups are absent in phosphatidylethanolamine derivatives such as the dietherPE, the dipole potential is not affected by arbutin, with a small decrease in the area. The effect of arbutin on the dipole potential differs from that of sucrose, trehalose and phloretin and is congruent with previous results obtained by FTIR on its interaction with the CO groups. Arbutin binding is interpreted in terms of the exposure to water of the phosphate and carbonyl groups at the membrane interface of the different monolayers.     

Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.     

Metastable ripple phase of fully hydrated dipalmitoylphosphatidylcholine as studied by small angle x-ray scattering

Fully hydrated dipalmitoylphosphatidylcholine (DPPC) undergoes liquid crystalline to metastable P_β, phase transition in cooling. A small angle x-ray scattering study has been performed for obtaining further evidence about the structure of this phase. From a high-resolution observation of x-ray diffraction profiles, a distinct multipeak pattern has become obvious. Among them the (01) reflection in the secondary ripple structure is identified clearly. There are peaks assigned straightforwardly to (10) and (20) reflections in the primary ripple structure and peaks assigned to (10) and (20) reflections in the secondary ripple structure. Therefore the multipeak pattern is due to superposition of the reflections cause by the primary and secondary ripple structures. The lattice parameters are estimated as follows: for the primary ripple structure a = 7.09 nm, b = 13.64 nm, and γ = 95°, and for the secondary ripple structure a = 8.2 nm, b = 26.6 nm, and γ = 90°. The lattice parameters thus obtained for the secondary ripple structure are not conclusive, however. The hydrocarbon chains in the primary ripple structure have been reported as being tilted against the bilayer plane and, on the other hand, the hydrocarbon chains in the secondary ripple structure are likely to be perpendicular to the bilayer plane. This fact seems to be related to a sequential mechanism of phase transitions. On heating from the L_β, phase where the hydrocarbon chains are tilted the primary ripple structure having tilted hydrocarbon chains takes place and on cooling from the L_α phase where the hydrocarbon chains are not tilted the secondary ripple structure with untilted chains tends to be stabilized. It appears that the truly metastable ripple phase is expressed by the second ripple structure although in the course of the actual cooling transition both the secondary and primary ripple structures form and coexist.     

ESR center line shifts during phase transitions of spin-labeled dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidyl ethanolamine

The position H_o of the ESR central absorption maximum of a spin-labeled phospholipid dispersion was recorded in the manner of a g-factor; γ=hν/βH_o. The point H_o corresponds to the center line zero crossing point of the first derivative ESR spectrum. A 5-doxyl derivative of stearic acid was incorporated into dispersions of dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidyl ethanolamine formed either by shaking a lipid film in the presence of buffered saline or by dialysis of label and lipid in 2-chloroethanol against buffered saline. It was found that during the gel to liquid crystal phase transition, the decrease in the order parameter, S, ranged from about .045 to .06 and the increase in the parameter γ ranged from about .0001 to .0002 depending upon the lipid and preparation method. In all cases examined, the changes could be associated with the lipid phase transition. A simple model based on rapid anisotropic motion indicates that about a 38% decrease in line width accounts for the observed shift in γ.     

Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle x-ray scattering

To clarify mechanisms of folding and unfolding of proteins, many studies of thermal denaturation of proteins have been carried out at low protein concentrations because in many cases thermal denaturation accompanies a great tendency of aggregation. As small-angle x-ray scattering (SAXS) measurements are liable to use low-concentration solutions of proteins to avoid aggregation, SAXS has been regarded as very difficult to observe detailed features of thermal structural transitions such as intramolecular structural changes. By using synchrotron radiation SAXS, we have found that the presence of repulsive interparticle interaction between proteins can maintain solute particles separately to prevent further aggregation in thermal denaturation processes and that under such conditions the thermal structural transition of hen egg-white lysozyme (HEWL) holds high reversibility even at 5% w/v HEWL below pH approximately 5. Because of the use of the high concentration of the solutions, the scattering data has enough high-statistical accuracy to discuss the thermal structural transition depending on the structural hierarchy. Thus, the tertiary structural change of HEWL starts from mostly the onset temperature determined by the differential scanning calorimetry measurement, which accompanies a large heat absorption, whereas the intramolecular structural change, corresponding to the interdomain correlation and polypeptide chain arrangement, starts much prior to the above main transition. The present finding of the reversible thermal structural transitions at the high protein concentration is expected to enable us to analyze multiplicity of folding and unfolding processes of proteins in thermal structural transitions.