Near-ultraviolet Circular Dichroism Spectra Research Articles

Correlation Between the Water Solubility and Secondary Structure of Tilapia-Soybean Protein Co-Precipitates.

The secondary structure of a protein has been identified to be a crucial indicator that governs its water solubility. Tilapia protein isolate (TPI), soybean protein isolate (SPI), and tilapia-soybean protein co-precipitates (TSPC3:1, TSPC2:1, TSPC1:1, TSPC1:2, and TSPC1:3) were prepared by mixing tilapia meat and soybean meal at different mass ratios. The results demonstrated that the water solubility of TSPCs was significantly greater than that of TPI (p <0.05). The changes in ultraviolet–visible and near-ultraviolet circular dichroism spectra indicated that the local structure of TSPCs was different from that of TPI and SPI. Fourier transform infrared Spectroscopy revealed the co-existence of TPI and SPI structures in TSPCs. The secondary structures of TSPCs were predominantly α-helix and β-sheet. TSPC1:1 was unique compared to the other TSPCs. In addition, there was a good correlation between the water solubility and secondary structure of TSPCs, in which the correlation coefficients of α-helix and β-sheet were −0.964 (p <0.01) and 0.743, respectively. TSPCs displayed lower α-helix contents and higher β-sheet contents compared to TPI, which resulted in a significant increase in their water solubility. Our findings could provide insight into the structure–function relationship of food proteins, thus creating more opportunities to develop innovative applications for mixed proteins.

Effects of Disulfide Bond Reduction on the Conformation and Trypsin/Chymotrypsin Inhibitor Activity of Soybean Bowman-Birk Inhibitor.

Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors (BBIs). In this study, highly purified BBI-A (approximately 96%) was obtained from soybean whey at the 0.1 g level by the complex coacervation method. BBI-A has seven disulfide bonds (SS) and no sulfhydryl group and exhibits trypsin inhibitor activity (TIA) and chymotrypsin inhibitor activity (CIA). The X-ray structure has shown that BBI-A has five exposed SS and two buried SS. Because of steric hindrance, it was reasonable to consider that dithiothreitol first attacks the five exposed SS and then the two buried SS, which was supported by the results that SS reduction with dithiothreitol could be divided into quick and slow stages, and the critical point was close to 5/7. The effects of SS reduction on TIA and CIA could be divided into three stages: when one exposed SS was reduced, both TIA and CIA decreased to approximately 60%; with further reduction of exposed SS, CIA gradually decreased to 8% and TIA gradually decreased to 26%; with further reduction of buried SS, CIA gradually decreased to 2% and TIA slightly decreased to 24%. Far-ultraviolet (far-UV) circular dichroism (CD) spectra showed that the secondary structure of BBI-A was slightly changed, whereas near-ultraviolet (near-UV) CD spectra showed that the conformation of BBI-A was substantially changed after the five exposed SS were reduced; further reduction of buried SS affected the conformation to some extent. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from a C8 column showed the same trend as near-UV CD spectra. BBI-A has a structural peculiarity in that two hydrophobic patches are exposed to the exterior (in contrast to typical soluble proteins), which was attributed to the seven SS by some researchers. These results support the hypothesis that hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core occurred after the reduction of SS in BBI-A.

A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue‐type plasminogen activator

A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex. To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue-type plasminogen activator (t-PA). The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS-PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP-mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis. SP enhanced the activation of plasminogen by t-PA. The catalytic efficiency (k(cat)/K(m)) of Glu-plasminogen activation by t-PA was 11.4-fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C-terminal of the plasminogen B region. Near-ultraviolet CD spectra of the complex between SP and Glu-plasminogen significantly differed from those of Glu-plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose-dependent manner. However, SP did not cause recanalization in t-PA gene-deficient mice. SP bound to the B region and promoted the activation of plasminogen by t-PA, and then induced effective thrombolysis.

Crystal Structure and Trimer-Monomer Transition of N-Terminal Domain of EhCaBP1 from Entamoeba histolytica

EhCaBP1 is a well-characterized calcium binding protein from Entamoeba histolytica with four canonical EF-hand motifs. The crystal structure of EhCaBP1 reveals the trimeric organization of N-terminal domain. The solution structure obtained at pH 6.0 indicated its monomeric nature, similar to that of calmodulin. Recent domain-wise studies showed clearly that the N-terminal domain of EhCaBP1 is capable of performing most of the functions of the full-length protein. Additionally, the mode of target binding in the trimer is similar to that found in calmodulin. To study the dynamic nature of this protein and further validate the trimerization of N-terminal domain at physiological conditions, the crystal structure of N-terminal domain was determined at 2.5 Å resolution. The final structure consists of EF-1 and EF-2 motifs separated by a long straight helix as seen in the full-length protein. The spectroscopic and stability studies, like far and near-ultraviolet circular dichroism spectra, intrinsic and extrinsic fluorescence spectra, acrylamide quenching, thermal denaturation, and dynamic light scattering, provided clear evidence for a conversion from trimeric state to monomeric state. As the pH was lowered from the physiological pH, a dynamic trimer-monomer transition was observed. The trimeric state and monomeric state observed in spectroscopic studies may represent the x-ray and NMR structures of the EhCaBP1. At pH 6.0, the endogenous kinase activation function was almost lost, indicating that the monomeric state of the protein, where EF-hand motifs are far apart, is not a functional state.

Spectroscopic and functional characterization of &amp;lt;italic&amp;gt;Lampyris turkestanicus&amp;lt;/italic&amp;gt; luciferase: a comparative study

Functional expression and spectroscopic analysis of luciferases from Lampyris turkestanicus and Photinus pyralis were carried out. cDNA encoding L. turkestanicus luciferase was isolated by reverse transcription-polymerase chain reaction, cloned, and functionally expressed in Escherichia coli. The luciferases were purified to homogeneity using Ni-nitrilotriacetic acid Sepharose, and kinetic properties of luciferase from L. turkestanicus were compared with that from P. pyralis. Amino acid differences in its primary structures in relation to P. pyralis luciferase brought about changes in the kinetic properties of the enzyme as evidenced by substantial lowering of Km for ATP, increased light decay time, and decreased thermostability. Luciferase from L. turkestanicus was used to carry out Michaelis-Menten kinetics with a Km of 95.5 muM for ATP and 20 muM for luciferin. Maximum activity was recorded at pH 8.5, so it might be a suitable reporter for microbial screening at alkaline pH. Tryptophan fluorescence for P. pyralis luciferase was higher than L. turkestanicus luciferase. Substitution of some residues in L. turkestanicus luciferase appears to change the kinetic properties by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by intrinsic and extrinsic fluorescence, Fourier transform infrared spectroscopy, and near-ultraviolet circular dichroism spectra.

Resolving near-ultraviolet circular dichroism spectra of single trp mutants in tear lipocalin

Near-ultraviolet circular dichroism (near-UV CD) spectra of tryptophan residues in proteins are complicated because the line shapes are derived from the overlap of both the 1L a and the 1L b electronic bands that vary independently. Contributing to this complexity, tryptophan near-UV CD spectra differ in the relative amplitude of the 0-0 vibronic band compared to the rest of the 1L b spectrum, an inherent feature that may result in poor fitting. To resolve this problem, a computer program that incorporated the separation of the 0-0 transition of 1L b component from the rest of the 1L b was written in LabVIEW and its amplitude was allowed to vary independently. This method showed dramatically improved fitting of 1L a and 1L b components in the near-UV CD tryptophan spectra in tear lipocalin mutants featuring low intensity of the 0-0 1L b component. Side chain dynamic characteristics (mobility and accessibility to the solvent) identified from different spectroscopic techniques were related to differences in Trp near-UV CD spectra. This method is broadly applicable to different types of Trp near-UV CD spectra.

Effect of a zwitterionic surfactant (HPS) on the conformation and hemolytic activity of St I and St II, two isotoxins purified from Stichodactyla helianthus.

N-hexadecyl-N-N'-dimethyl-3-ammonio-1-propane-sulfonate (BPS) is a zwitterionic surfactant that readily binds to sticholysins I and II, two sea toxins isolated from Stichodactyla helianthus. The binding constants, evaluated from changes in fluorescence intensities elicited by the surfactant, are approximately 0.5-0.7 microM(-1). The binding of the surfactant changes the conformation of the tertiary protein, without significant changes in its secondary structure, as reported from far-ultraviolet circular dichroism spectra. The changes elicited by HPS lead to loss of the native conformation (as reported from near-ultraviolet circular dichroism spectra) and to a shift of the intrinsic protein fluorescence toward longer wavelengths, an increase in fluorescence intensities and lifetimes, and a faster quenching by acrylamide. All these changes are indicative of a more expanded tertiary conformation. Despite this, the toxins fully retain their hemolytic activities, indicating that spectroscopic changes can be poor predictors of toxin activity.

β-Lactoglobulin Molten Globule Induced by High Pressure

Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Spectroscopic and Thermodynamic Characterization of the Transcription Antitermination Factor NusE and Its Interaction with NusB from Mycobacterium tuberculosis

N-utilizing proteins (Nus) form a complex involved in the regulation of rRNA biosynthesis in enteric bacteria by modulating the efficiency of transcriptional termination [Nodwell, J. R., and Greenblatt, J. (1993) Cell 72, 261-268]. The protein NusE (identical to the protein S10 of the small ribosomal subunit) from the pathogenic mycobacterium M. tuberculosis has been cloned and overexpressed in Escherichia coli. The pure protein has been characterized by circular dichroism, ultracentrifugation, NMR, and binding to NusB. The near-ultraviolet circular dichroism spectrum of this protein suggests that it has a moderate (ca. 12-16%) alpha-helical content at 30 degrees C. The protein undergoes cold denaturation, with a temperature of maximum stability near 40 degrees C, implying a substantial heat capacity difference between the folded and unfolded states. The sedimentation equilibrium and velocity data indicate that the protein is monomeric and expanded in solution. NMR spectroscopy shows that there is no significant tertiary structure, and confirms the low secondary structure content at low temperatures. Furthermore, there was evidence for more structure at 30 degrees C than at 10 degrees C. Well-defined shifts in peaks in the HSQC spectrum of (15)N labeled NusE/NusB when the unlabeled counterpart was added at approximately stoichiometric concentrations showed the formation of a NusE-NusB complex in the absence of RNA. The far-UV CD and ultracentrifuge experiments, however, indicated relatively weak binding. Isothermal titration calorimetry showed the binding was weak and endothermic at 15 degrees C, with a total DeltaH of > or =10 kcal/mol. This weak binding is consistent with a small interaction interface and lack of large conformational rearrangements in the predominantly unfolded NusE protein. The conformational flexibility of NusE may be important for its roles in both the ribosome and antitermination complexes.

The acid-induced folded state of Sac7d is the native state.

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.

Exploring the conformational properties of the sequence space between two proteins with different folds: an experimental study

We have examined the conformational properties of 27 polypeptides whose sequences are hybrids of two natural protein domains with 8 % sequence identity and different structures. One of the natural sequences (spectrin SH3 domain) was progressively mutated to get closer to the other sequence (protein G B1 domain), with the only constraint of maintaining the residues at the hydrophobic core. Only two of the mutants are folded, each of them having a large sequence identity with one of the two natural proteins. The rest of the mutants display a wide range of structural properties, but they lack a well-defined three-dimensional structure, a result that is not recognized by computational tools commonly used to evaluate the reliability of structural models. Interestingly, some of the mutants exhibit cooperative thermal denaturation curves and a signal in the near-ultraviolet circular dichroism spectra, both typical features of folded proteins. However, they do not have a well-dispersed nuclear magnetic resonance spectrum indicative of a defined tertiary structure. The results obtained here show that both the hydrophobic core residues and the surface residues are important in determining the structure of the proteins, and suggest that the appearance of a completely new fold from an existing one is unlikely to occur by evolution through a route of folded intermediate sequences.

The Binding of Azide to Copper‐Containing and Cobalt‐Containing Forms of Hemocyanin from the Mediterranean Crab Carcinus Aestuarii

To establish the competence of the active site of hemocyanin to acquire diverse coordination geometries, the binding of azide to three forms of a crab hemocyanin, the dinuclear cupric or met-hemocyanin, the mononuclear cupric or met-apo-hemocyanin, and the mononuclear Co(II)-substituted derivative has been studied by near-ultraviolet circular dichroism and EPR spectroscopies. The near-ultraviolet circular dichroism spectra of the various derivatives present qualitatively similar features, namely a negative peak around 335 nm in the case of the two copper-containing derivatives and a three-component pattern with the Co(II) derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optical activity is observed with all protein samples. The characteristic CD features, attributable to N(imidazole)-to-metal and to OH -to-metal charge-transfer transitions, are strongly affected by azide binding. In particular, the intensity of the negative band exhibited by the two copper-containing protein forms decreases with the onset of a new negative feature with maximum around 400 nm diagnostic for azide-to-Cu(II) charge-transfer transitions. The visible region is affected as well, indicating that changes in the coordination sphere of copper take place. The affinity for azide of the different protein forms is higher at low pH. EPR measurements on the paramagnetic met-apo-hemocyanin derivative as a function of pH demonstrate heterogeneity in the coordination environment at low pH. In the presence of azide an increase of rhombic distortion of the EPR spectra is observed and on the basis of the identified sets of copper hyperfine features in the course of azide titration experiments two different azide bound forms of met-apo-hemocyanin can be detected. The CD and EPR data at the different pH values are consistent with a reaction scheme in which azide replaces a fourth ligand in the metal-coordination sphere, identified as a water or hydroxide molecule.

Molten globule state of equine β‐Lactoglobulin

The acid-unfolded state of equine β-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine β-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedimentation experiments, and it has the exposed hydrophobic surface as indicated by its tendency to aggregate. All of these characteristics indicate that the acid-unfolded state of equine β-lactoglobulin is a molten globule state. The α helix content in the acid-unfolded state, which has been estimated from the circular dichroism spectrum, is larger than that in the native state, suggesting the presence of nonnative α helices in the molten globule state. This result suggests the generality of the intermediate with nonnative α helices during the folding of proteins having the β-clam fold. © 1997 Wiley-Liss Inc.

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.

Modulation of the oxidation-reduction potential of the flavin in lipoamide dehydrogenase from Escherichia coli by alteration of a nearby charged residue, K53R.

The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD [Pai & Schulz (1983) J. Biol. Chem. 258, 1752]. A conserved lysine residue occupies a similar position in lipoamide dehydrogenase. This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R. The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD+ are profoundly affected by the change. K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate. The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pKa = 7.0. Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum and indicates an apparent Kd = 7.0 microM at pH 7.6. The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD+. K53R is extensively reduced (mostly EH4) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH2). The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a ribonuclease S refolding intermediate

D uring ribonuclease S (RN ase S) refolding, two peptide fragm ents recognize each other, and bind to g eth er to form a refolding in term ediate which slowly converts to th e nativ e state. We have characterized this refolding interm ediate using absorbance, circular dichroism (CD), and nuclear m agnetic resonance (NMR) spectroscopies. These techniques reveal significant am ounts of bo th secondary and te rtia ry stru ctu re ; the in term ediate differs from a m olten globule in being packed and native-like, b u t it resembles a m olten globule in having no near-ultraviolet (UV) CD spectrum . Final refolding is slow and accom panies proline isom erization. The results show th a t a t least two separate stages are observed in the form ation of the te rtia ry stru ctu re of RNaseS.

Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region

Far-ultraviolet circular dichroism and tryptophan fluorescence measurements showed that the reversible unfolding of the cysteine proteinase inhibitor, chicken cystatin, by guanidinium chloride is a two-step process with transition midpoints at ≈3.4 and ≈5.4 M denaturant. The partially unfolded intermediate had both far- and near-ultraviolet circular dichroism and fluorescence emission spectra comparable to those of the native protein. The largely retained tertiary structure suggests that the intermediate represents a species in which a separate region of lower stability has been unfolded, rather than an intermediate of the ‘molten globule’ type. Such a structurally independent region is apparent in the three-dimensional structure of the inhibitor.

Stabilization of the Z′ form of poly(dGdC):poly(dGdC) in solution by multivalent ions relates to the Z¶form in crystals

Both multivalent ions and 85% ethanol are required to produce the original Z' form of poly(dGdC):poly(dGdC) in solution. When multivalent impurities are removed by dialysis against 0.5 M NaCl and 1 mM EDTA, the circular dichroism retains features of the standard Z form. Addition of Ca+2 nearly reverses this effect. Analysis by singular value decomposition of near-ultraviolet circular dichroism spectra collected during titrations of polynucleotide in 60% ethanol with multivalent ions reveal that, at concentrations below .5 per nucleotide, they stabilize Z'-like forms in a two-state equilibrium with the Z form. Differences among the Z' spectra produced by the different ions suggest that at least three families of Z' structures exist. Furthermore, comparison with crystal data indicates that the Z' form in solution is related to the ZII form in crystals.

Conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes.

The conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes were studied by using near- and far-ultraviolet circular dichroism. The far-ultraviolet spectrum of MIP26 solubilized with octyl beta-D-glucopyranoside indicates an alpha-helical content of approximately 50% and a beta-structure content of approximately 20%. A detergent-free membrane suspension of MIP26 produced a typically distorted far-ultraviolet spectrum which was caused by differential light scattering and absorption flattening. However, decreasing the size of the membrane fragments by sonication produced a far-ultraviolet spectrum free of distortion, and with a rotatory strength profile similar to that obtained for MIP26 solubilized with octyl beta-D-glucopyranoside. This implies similar secondary structure properties for the protein in both the suspension and the sugar detergent. The cleavage of MIP26 with Staphylococcus aureus protease, which results in removal of a 5-kilodalton peptide and which mimics the age-dependent posttranslational changes that take place in the lens, did not significantly affect the conformation of the core protein as judged by the near-ultraviolet circular dichroism spectra.