Effect of the carbohydrate moiety on the secondary structure of beta 2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins

By use of six highly purified exoglycosidases with well-defined specificity, the oligosaccharide units of human plasma beta 2-glycoprotein I (beta 2I) were modified by sequential enzymatic degradation. The released monosaccharides (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoprotein (gp) derivatives were determined. The gp was found to be both partially sialylated and galactosylated. These findings which are in agreement with earlier reports suggest that the carbohydrate moiety of beta 2I possesses more bi- than tri-antennas, probably three of the former and two of the latter carbohydrate units. Circular dichroic (CD) spectra of native beta 2I and its derivatives were measured in aqueous buffer and 2-chloroethanol (2-CE). Analysis of these spectra for elements of secondary structure showed beta 2I and most of the derivatives to contain predominantly beta-sheet and beta-turn structures. The lack of alpha-helical structures in aqueous buffer was noted. Removal of a large portion of the carbohydrate moiety did not alter the CD spectra or secondary structure of beta 2I in either aqueous buffer or in 2-CE. However, after enzymatic removal of approximately 96% of the carbohydrate moiety, large significant changes in the spectra and secondary structures were observed. In aqueous buffer a shift in the wavelength minimum occurred, accompanied by an increase in the magnitude of the molar ellipticity and the amount of beta-turn, with a reduction in random coil. One-third of the amino acids which were originally in random coil conformation assumed beta-turns after removal of 96% of the carbohydrate moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

β2-Strand of salivary S cystatins: A “chemeleon sequence”

Secondary structure prediction of salivary cystatins S, SA, and SN carried out by several methods label the 39-58 sequence (β2-strand) as predominantly α-helical. The helical propensity of a peptide corresponding to β2-strand of salivary SA cystatin analyzed by CD display high helical propensity in aqueous solution, whereas peptides matching the β2-strand amino acid sequence of cystatins S and SN, display random coil conformation in aqueous solution but acquire α-helical conformation in the presence of trifluoroethanol (TFE). Moreover molecular dynamics simulation performed on the homology modeling of cystatin SA constructed on the basis of recently determined three-dimensional structure of salivary cystatin D, suggests that cystatin SA does not significantly deviate from the starting structure over the course of the simulation. The results obtained indicate that the β2-strand of salivary S cystatins has high helical propensity when isolated from native protein and acquire the final β structure by interaction with the rest of the polypeptide chain.     

Solvent denaturation of proteins as observed by resolution-enhanced Fourier transform infrared spectroscopy

Fourier self-deconvolution of Fourier transform infrared (FTIR) spectra and second derivative FTIR spectroscopy were applied to study solvent-induced conformational changes in globular proteins. For beta-lactoglobulin a total of three different denatured forms were identified in alkaline solution and in aqueous methanol-d1 and isopropanol-d1. In isopropanol-d1 solution a new conformation was identified which appears to resemble, but is not identical with, the beta-structure of native proteins. This conformation is characterized by absorption bands around 1615-1618 and 1684-1688 cm-1, and is also observed for concanavalin A and chymotrypsinogen A in aqueous isopropanol-d1 solution.     

Conformational stability of P22 tailspike proteins carrying temperature-sensitive folding mutations

The thermostable tailspike endorhamnosidase of Salmonella phage P22 provides a model system for comparing the role of amino acid sequences in determining the intracellular folding pathway with their role in stabilizing the mature structural protein. Complete Raman band assignments are given here for the native form of the tailspike trimer in aqueous solution. Once correctly folded and assembled, the wild-type and two well-characterized mutant proteins, tsfIle258----Leu and tsfGly323----Asp, exhibit the same secondary structure in solution, consisting predominantly of beta-strand (56 +/- 5%) and turns (17 +/- 2%). Raman bands that are sensitive indicators of hydrogen-bonding interactions of tyrosine (phenolic OH) and tryptophan (indole NH) are unchanged between 30 and 80 degrees C in both wild type and tsf mutants. Similarly, Raman bands that are sensitive to changes in the hydrophobic environment of nonpolar side chains exhibit no significant temperature dependence in wild type and tsf mutants. In contrast, these conformational features are greatly altered by chemical denaturation of the tailspike with lithium halide and guanidine hydrochloride. In the chemically denatured tailspike, the beta-strand structure is substantially converted to irregular or "random coil" conformation. These findings confirm conclusions from physiological studies that the three-dimensional structures of the tsf mutants, once stabilized at permissive temperatures, are equivalent to the native structure of the wild type, and this structure is maintained at temperatures far above those that block the folding of the chain into the final native conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser Raman spectroscopy of calf bone Gla protein

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of alpha-helical, antiparallel beta-sheet, and random-coil regions in all four forms of bone Gla protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased alpha-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm-1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca2+ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca2+ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.     

Vibrational circular dichroism studies of epidermal growth factor and basic fibroblast growth factor.

Vibrational circular dichroism (VCD) studies are reported for two unrelated recombinant growth factor proteins: epidermal growth factor and basic fibroblast growth factor (bFGF). NMR, electronic CD, and bFGF X-ray studies indicate that these two proteins are primarily composed of beta-sheet and loop secondary structure elements with no detectable alpha-helices. Two reports on solution conformation of these proteins using FTIR absorption spectroscopy with subsequent resolution enhancement confirmed the presence of a large fraction of a beta-sheet conformation but in addition indicated the presence of large absorption bands in the 1650-1656 cm-1 region, which are typically assigned to alpha-helices. The VCD spectra of both proteins have band shapes that strongly resemble those of other high beta-sheet fraction proteins, such as the trypsin family of proteins. Quantitative analysis of the VCD spectra also indicates that these proteins are predominantly in beta-sheet and extended ("other") conformations with very little alpha-helix fraction. These results agree with the CD interpretation and affirm that the FTIR peaks in the region 1650-1656 cm-1 can be assigned to loops. This study provides an example of the limitations of using FTIR frequencies alone for examination of protein secondary structure.     

The conformation of bombesin in solution as determined by two-dimensional 1H-NMR techniques

The 1H nuclear magnetic resonance spectrum of the tetradecapeptide, bombesin, has been assigned in (2H6)dimethyl sulphoxide solution and aqueous solution using two-dimensional techniques. The chemical shifts in both solvents indicate that the molecule has little secondary structure and adopts a random coil conformation. A comparison is made between the spectra of various smaller bombesin fragments and the intact polypeptide.     

Raman spectroscopic evidence for a disulfide bridge in calf gamma II crystallin

Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical gamma II crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of gamma II as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-1), the native form shows a line near 511 cm-1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-1. In every other spectral region, both native and reduced gamma II forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.     

Crystal and solution structures of the B-DNA dodecamer d(CGCAAATTTGCG) probed by Raman spectroscopy: heterogeneity in the crystal structure does not persist in the solution structure

The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Revisiting the conformation of xanthan and the effect of industrially relevant treatments

The structure and conformation of xanthan in aqueous solution following various processing treatments typically encountered in its application were investigated in this study. Treatments such as heating, autoclaving, high pressure homogenisation and irradiation were subjected to the same sample. Parameters such as weight average molecular weight (M(w)), polydispersity index, root mean square radius of gyration, intrinsic viscosity and Huggins constant were used to monitor the effect of these treatments. Additionally, we have quantified the mass recovery of samples examined by gel permeation chromatography and light scattering to properly account for all fractions present in xanthan solutions. Atomic force microscopy (AFM) images together with height measurements confirmed that xanthan conformation is double helical ordered renatured state (pre-heat treated by the manufacturer) in dilute solution conditions and random coil conformation in very dilute solution. The ordered (renatured) conformation is shown to have partially molten double helix, with more flexibility than the perfectly ordered native double helix. Heat treatment for 2h at 85°C reduces the M(w) of xanthan to half its initial value, and mass recovery measurements indicate that it completely overcomes its associative nature. Thermally treated xanthan solution in the dilute region leads to an order-disorder transition, as determined by contour length per unit mass. Similarly, irradiation of xanthan solution results in an order-disorder transition together with the production of single strand low molecular weight molecules. Autoclaving and high pressure homogenisation treatments cause degradation of xanthan. The results from treated xanthan solutions following high pressure homogenisation and irradiation confirm that xanthan does not reassociate. A revised summary of xanthan conformation in solution together with schematic models following the various treatments are proposed.     

Conformational transition and liquid crystalline state of regenerated silk fibroin in water

The conformational transition of molecular chains of regenerated silk fibroin (SF) aqueous solution is systematically investigated by circular dichroism, Raman, IR, and UV-vis spectroscopies. It is found that an initial random coil conformation of the SF can be readily changed into an ordered beta-sheet structure by optimizing the solution conditions, such as the SF concentration, pH, temperature, or metal-ion content. Circular dichroic spectra quantitatively confirm a steadily decreased content of the random coil conformation but a significantly increased beta-sheet content after an ultrasonic or extruding treatment. Furthermore, the extrusion is more powerful to achieve high beta-sheet content than the ultrasonic. It is interesting that the polarized optical micrographs of the SF aqueous solution extruded by injection illustrate the formation and existence of liquid crystalline state. A study of extrusion in vitro could be used as a model system to understand the natural silk spinning process in silkworm.     

Circular dichroism studies of the fatty acid synthetase complex from the insect Ceratitis capitata

Circular dichroism spectra of the native fatty acid synthetase complex from the insect Ceratitis capitata and of the lipidated and cholate- and SDS-treated enzyme have been obtained. Native enzyme has a calculated structure of 43% α-helix, 23% β structure and 31% random coil. Lipidation and cholate-treatment did not modify the structure of the enzyme complex whereas the SDS-treatment changed the native conformation into a structure based on 42.8% α-helix, 8.4% β structure and 48.8% random coil. These data are interpreted in terms of both the enzyme activity and the quaternary structure of the complex.     

Triple helical polysaccharide-induced good dispersion of silver nanoparticles in water

Silver nanoparticles were constructed by using triple helical polysaccharide (lentinan) dissolved in water as matrix for the first time. The structure, morphology, and size of the nanocomposites in the polysaccharide aqueous solutions were investigated with UV-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), and dynamic laser light scattering (DLS). The results revealed that the silver nanoparticles were attached to the polysaccharide chains through the strong noncovalent interactions, leading to the good dispersion of silver nanoparticles with mean radius of 6 nm in water. The silver nanoparticles were stable in the lentinan aqueous solution for 9 months. However, with an addition of NaOH, the polysaccharide with the imperfect helical structure broken partially by NaOH could aggregate in the alkali aqueous solution. The aggregation of the lentinan-bonded silver nanoparticles increased with an increase in the NaOH concentration, whereas the size of the silver nanoparticles barely changed, further confirming that the Ag nanoparticles were stable in this system. The aggregation was related to the conformation transition of the polysaccharide from the triple helix to random coil in the solution. A new method to detect the aggregates and aggregation rate was established according to the intensity of the maximum absorption peaks of the polysaccharide labeled by Ag nanoparticles in the UV spectrum.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Assemblages of prion fragments: novel model systems for understanding amyloid toxicity

We report the conformational and toxic properties of two novel fibril-forming prion amyloid sequences, GAVVGGLG (PrP(119-126)) and VVGGLGG (PrP(121-127)). The conformational preferences of these fragments were studied in differing microenvironments of TFE/water mixtures and SDS solution. Interestingly, with an increase in TFE concentration, PrP(119-126) showed a helical conformational propensity, whereas PrP(121-127) adopted a more random coil structure. In 5% SDS, PrP(119-126) showed more alpha-helical content than in TFE solution, and PrP(121-127) exhibited a predominantly random coil conformation. However, both peptides took a random coil conformation in water, and over time the random coil transformed into a beta-sheet structure with a significant percentage of helical conformation and beta-turn structure in PrP(119-126) and PrP(121-127), respectively, as observed with CD spectroscopy. The aged fibrils of PrP(119-126) were insoluble in SDS, and PrP(121-127) was extractable with SDS solution. These fibrils were characterized by transmission electron microscopy. Both PrP(119-126) and PrP(121-127) formed stable monolayer's consisting of multimeric assemblages at the air-water interface. Monomeric PrP(119-126) was more toxic to astrocytes than the control Abeta peptide; however, the fibrillar form of PrP(119-126) was less toxic to astrocytes. PrP(121-127) elicited moderate toxicity in both soluble and fibrillar forms on astrocytes. Furthermore, quenching experiments using acroyl-labeled PrP(119-126) and PrP(121-127) with eosin-labeled synaptosomal membrane revealed that these prion fragments bind to anion-exchange protein. The binding of PrP(119-126) and PrP(121-127) with a membrane microdomain (lipid raft) was also analyzed using pyrenated derivatives. We conclude that the formation of PrP(119-126) and PrP(121-127) fibrils is a concentration-dependent process that involves coil to sheet conversion with aging. PrP(119-126), the sequence with intrinsic helical propensity, is more toxic in monomer form, and the fibril formation in this case seems to be protective to cells. For PrP(121-127), the SDS-soluble fibrils are more cytotoxic, indicating that a higher order assemblage structure is required for cytotoxic activity of this peptide.     

The C-terminus of bacteriorhodopsin is a random coil

The 21 amino acids which can be selectively removed from the carboxyl terminus of bacteriorhodopsin by proteolytic treatment are disordered in 2-dimensional arrays of the protein present in purple membranes. This C-terminal portion of the molecule may be involved in the efficiency and rate of light-driven proton uptake, although its presence is not required for pumping activity. In this study, the secondary structure of the C-terminus of bacteriorhodopsin has been determined by examining circular dichroism (CD) difference spectra derived from native and digested samples. In low ionic strength media, this part of the molecule appears to form a random coil-like structure. To examine if this structure is related to the structure found under the high ionic strength condition present in halobacteria, the CD spectra of native purple membranes in water and in 4 M salt solutions were compared. They were found to be identical, suggesting the conformation of the C-terminus in vivo may also be a random coil.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

Solution properties of gum exudates from Sterculia urens (Karaya gum)

Dilute solution properties of Karaya gum from Sterculia urens were studied using size-exclusion chromatography, static and dynamic light scattering and viscosity experiments in 0·1 aq. NaCl.

Solubility in water was found to be strongly dependent on the degree of acetylation. The native acetylated Karaya gum assumes a rather compact and branched conformation in aqueous solution, as evidenced by the low values of power-law exponents. In contrast, the fully deacetylated Karaya gum assumes a more expanded conformation and behaves as a random coil.     

Protein structure by Fourier transform infrared spectroscopy: second derivative spectra

Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein.