The crystal structure of cholesteryl octanoate

Crystals of cholesteryl octanoate (C₃₅H₆₀O₂) are monoclinic, space group P2₁, with $\text{a = 12.80(3), b = 9.20(2), c = 14.12(3)}\text{A}\text{?}$, β = 93.81(3)° and 2 molecules per unit cell. The structure has been determined by Patterson rotation and translation methods from the X-ray intensities (Mo-Kα radiation) of 1320 reflections ($\text{sin}\text{θ/λ < 0.59}\text{A}\text{?}{}^{\mathrm{?1}}$) measured with a diffractometer. Refinement by block diagonal least squares and Fourier methods gave R = 0.096. The molecules are arranged in monolayers with their long axes antiparallel and severely tilted (28°). There is a close packing of cholesteryls within the monolayers, but the octanoate chains which form the layer interface regions are conformationally and thermally disordered. The crystal structure is quite different from that of cholesteryl nonanoate, as expected from the discontinuity in thermodynamic properties and phase behaviour which occurs at this point in the homologous series.     

The crystal structure of cholesteryl 17-bromoheptadecanoate

Crystals of cholesteryl-17-bromoheptadecanoate (C₄₄H₇₇BrO₂) are monoclinic (P2₁) with a = 7.663(2), b = 10.311(5), c = 55.96(2) A and β = 103.10(3°). These are two molecules in the asymmetric unit which have different conformations of the cholesterol side chain and about the ester bond. The molecules pack with regions of only steroid skeleta alternating with regions of hydrocarbon chains. Due to the packing requirements of the skeleta the carbon chains are forced into a hybrid type packing which contains features of the earlier known O⊥ and T∥ subcells. The subcell (HS1) is orthorhombic with a_s = 10.3, b_s = 7.5 and c_s = 2.54A. The molecular packing is such that the ω-bromine atoms do not continue the trans-carbon chains but adopt a gauche conformation.     

The crystal structure of cholesteryl dihydrogen phosphate

Crystals of cholesteryl dihydrogen phosphate grown from 1,4-dioxane solution are monoclinic, space group C2 with $\text{a = 24.40, b = 6.27, c = 40.86}\text{A}\text{?}\text{and}\text{β = 102.7°}$. The asymmetric unit contains two molecules of cholesteryl phosphate CP and one dioxane molecule of the solvent. The CP molecules pack tail to tail in a bilayer structure. Within the layer they are arranged in double rows with their phosphate groups linked to ribbons by hydrogen bonds. Laterally the double strands of phosphate groups are separated by rows of dioxane molecules. The dioxane serves as hydrogen bond acceptor and as a spacer molecule that compensates the differences in cross-sectional area of the cholesteryl residue (38.4 Ų and the phosphate group (24 Ų). In the cholesterol matrix the CP molecules joined to double rows have packing contact with the smooth side of their skeleta and interdigitate with their annular methyl groups with those of molecules of the adjacent double rows. The branched cholesteryl side chains facing the bilayer center are loosely packed and show considerable disorder and/or thermal motion.     

Spruce budworm antifreeze protein: changes in structure and dynamics at low temperature

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.     

Conformational changes in ovalbumin at acid pH

Several physicochemical parameters of ovalbumin were examined at acid pH. The intrinsic viscosity and far UV-CD spectrum at pH 2 did not differ from those at pH 7. But the near UV-CD spectrum, difference absorption spectrum around 250-320 nm, and fluorescence spectrum showed micro-environmental changes around the aromatic amino acid residues in acid solution. The reactivity of one of the four sulfhydryl groups with 2,2'-dithiodipyridine increased at pH below 5. The rate of denaturation by urea and that of surface tension decay were high in the low pH range. We concluded that at low pH (around 2), ovalbumin molecules kept their native globular conformation, but that their chain flexibility increased and they were very susceptible to denaturation. This state might be equivalent to the molten-globule state observed with some globular proteins in acidic region.     

Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with single- and double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 Å in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short β-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.     

Model phospholipid membranes affect the tertiary structure of holomyoglobin: Conformational changes at pH 6.2

The influence of model negatively charged membranes on the structure of sperm whale holomyoglobin at pH 6.2 has been investigated using a variety of techniques (far-UV and near-UV circular dichroism, tryptophan fluorescence, absorption spectroscopy, differential scanning microcalorimetry, and fast-performance liquid chromatography). It has been shown that, similarly to apomyoglobin, holomyoglobin in the presence of phospholipid vesicles undergoes a conformational transition from the native to the intermediate state, which is characterized by loss of the rigid tertiary structure and the native heme environment; at the same time, the content of -helical secondary structures remains virtually unchanged. The molar phospholipid/protein ratio required for this transition is higher than in the case of apomyoglobin. The properties of holomyoglobin in the presence of negatively charged membranes are largely similar to those of the molten globule state of its apo form in aqueous solution. A possible functional role of this novel non-native state of myoglobin in the cell is discussed.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 120–128.Original Russian Text Copyright © 2005 by Basova, Tiktopulo, Bychkova.     

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.     

Low temperature crystal structure of natural diosgenone

The molecular structure of diosgenone, a natural steroidal sapogenin, closely related to diosgenin and isolated from Solanum nudum, was solved by single crystal X-ray diffractometry at 120 K and refined by full-matrix least-squares to an agreement factor, R1 = 0.054. It crystallizes in the monoclinic space group P2(1), with a = 15.1870(4) A, b = 7.2710(2) A, c = 21.2840(6) A, beta = 99.251(1) degrees, and four molecules in the unit cell (Z = 4). The results constitute the first structural report on a steroidal sapogenin from the diosgenin group.     

Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.     

The crystal structure of cholesteryl dodecanoate: co-packing of steroid skeleta and hydrocarbon chains

The crystal structure of cholesteryl dodecanoate has been determined. The compound shows a co-packing of cholesterol skeleta and hydrocarbon chains. There are two molecules in the asymmetric unit both almost fully extended. The hydrocarbon chain axes are however somewhat bent in order to get a good close-packing side by side with the rigid cholesterol skeleta. The two non-symmetry related skeleta show different packing surroundings. One skeleton packs with both hydrocarbon chains and other skeleta while the other skeleton is completely surrounded by hydrocarbon chains. The latter packing is of particular interest as it is considered to indicate important packing principles in biological lipid bilayers.     

Conformational changes in single-strand DNA as a function of temperature by SANS

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5'-ATGCTGATGC-3', in sodium phosphate buffer solution at 10 degrees C temperature increments from 25 degrees C to 80 degrees C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25 degrees C to 43 degrees C, an expansion in the pitch direction with a slight decrease in the diameter from 43 degrees C to 53 degrees C, and finally a dramatic increase in the pitch and diameter from 53 degrees C to 80 degrees C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5 +/- 0.5 degrees C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 +/- 3 kJ mol(-1) that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 +/- 1 degrees C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature.     

Crystal structure of form II of cholesteryl palmitelaidate at 295 K

Form II for cholesteryl palmitelaidate (trans-9-hexadecenoate) (C43H74O2) is monoclinic P2(1) with a = 12.745(3), b = 9.006(2), c = 18.153(4) A, beta = 96.63 (2) degrees, Z = 2. The X-ray crystal structure of form II has been determined from 2506 reflections of which 2126 gave (F greater than 2 sigma). The data up to sin theta/lambda = 0.44A-1 (Dmin = 1.14 A) were measured with CuK alpha radiation from a sealed tube. These were supplemented up to sin theta/lambda = 0.52 A-1 (Dmin = 0.96 A) by measurements on the same crystal using a rotating anode X-ray source. The electron density was diffuse in the ester chain and the atoms of the cholesteryl tail were found to be disordered. The tail and the chain atoms were refined by restrained least squares methods to give R = 0.087 and Rw = 0.10 for reflections with F greater than 2 sigma. Crystal forms I and II represent two standard structure types already characterized for fatty acid esters of cholesterol. In form II, the ester chain is almost fully extended as is also the case for one of the two independent molecules (A) in form I. In form II, the chains pack loosely together for most of their length. M.s. amplitudes of thermal vibration for the chain C-atoms are almost uniform along the entire chain (approximately 0.25 A2 at 295 K). In form I, the proximal part of the A chain is surrounded by rigid cholesteryl groups. In this region, C-atom m.s. amplitudes are much reduced (approximately 0.10 A2) but they increase to about 0.5 A2 at the distal end of the chain where packing is very loose.     

Conformational changes in bovine lactoferrin induced by slow or fast temperature increases

Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70 degrees C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25 degrees C to 105 degrees C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.     

Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.     

Endosome-lysosome fusion at low temperature

Based on an initial study (Dunn, W. A., Hubbard, A. L., and Aronson, Jr., N. N. (1980) J. Biol. Chem. 255, 5971-5978), low temperature is often used to selectively inhibit fusion between endosomes and lysosomes. Here we have tried to characterize the nature of this inhibition. In addition to endocytic contents markers, we have used a covalent membrane marker to measure the interaction between endosomes and lysosomes over extended periods of time at low temperature. Mouse macrophage cells (P388D1) and human skin fibroblasts were enzymatically labeled with radioactive galactose to provide a covalent marker for plasma-membrane glycoconjugates. Subsequent endocytic membrane traffic for 24 h at 16 degrees C resulted in a significant transfer of membrane marker, as well as of endocytic contents marker, to high density lysosomes, as observed by subcellular fractionation. The kinetics of this transfer have been analyzed for macrophages using the membrane marker, horseradish peroxidase as fluid-phase, and iodinated acetyl low density lipoprotein as receptor-mediated endocytic contents marker. Transfer to lysosomes occurred only about 6 h after application of the respective marker at 16 degrees C. When transfer to lysosomes was initiated by 15 min preincubation at 37 degrees C, subsequent cooling to 16 degrees C did not inhibit ongoing transfer which continued with the same kinetics as when observed after the lag phase. These results show that low temperature delays an unidentified pre-fusion step, but does not inhibit endosome-lysosome fusion as such.     

Conformational changes in plastocyanin

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.