Interaction of insulin with a triblock copolymer of PEG-(fumaric-sebacic acids)-PEG: thermodynamic and spectroscopic studies

A comparative study on the interaction of (PEG-co-P(FA/SC)-co-PEG) triblock copolymer with bovine and human insulins was carried out using isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy. ITC data show that the copolymer has a low affinity for both proteins, with an association constant of about 7-9 x 10(3) M (-1). Results also show that binding is enthalpically driven, and disfavored by conformational entropy. CD spectroscopy studies reveal a small increase in the helical content and a decrease in beta-structure as well as random coil in both proteins. Acrylamide quenching experiments display reduced accessibility of tyrosines, while intrinsic fluorescence spectra show lower tyrosine emission. Furthermore, thermal unfolding experiments, studied by far-UV CD at 222 and 217 nm, demonstrate that upon interaction with the copolymer helix structure becomes less stable while the stability of beta-structure remains unchanged. Altogether, these observations indicate that (PEG-co-P(FA/SC)-co-PEG) triblock copolymer has similar effect(s) on both proteins.     

Tau paired helical filaments from Alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain

Tau protein, a neuronal microtubule-associated protein, forms insoluble fibers ("paired helical filaments") in Alzheimer's disease and other tauopathies. Conflicting views on the structure of the fibers have been proposed recently, ranging from mainly alpha-helical structure to mainly beta-sheet, or a mixture of mostly random coil and beta-sheet. We have addressed this issue by studying tau fibers immunopurified from Alzheimer brain tissue by a conformation-specific antibody and comparing them with fibers reassembled from recombinant tau or tau constructs in vitro, using a combination of electron microscopy and spectroscopic methods. Brain-derived fibers and reassembled fibers both exhibit a typical twisted appearance when examined by electron microscopy. The soluble tau protein is a natively unfolded protein dominated by random coil structure, whereas Alzheimer PHFs and reassembled fibers show a shift toward an increase in the level of beta-structure. The results support a model in which the repeat domain of tau (which lies within the core of PHFs) adopts an increasing level of beta-structure during aggregation, whereas the N- and C-terminal domains projecting away from the PHF core are mostly random coil.     

Evidence for an interaction between the membrane protein of a paramyxovirus and actin.

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.     

Secondary structural changes of non-reduced and reduced ribonuclease A in solutions of urea, guanidine hydrochloride and sodium dodecyl sulfate

The secondary structures of ribonuclease A (RNAase A) before and after reduction of the disulfide bridges and blockage of the thiol groups with iodoacetamide were examined in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate (SDS). The relative proportions of alpha-helix, beta-structure, and disordered structure were estimated by the curve-fitting method of circular dichroism (Chen, Y.H., Yang, J.T. and Chau, K.H. (1974) Biochemistry 13, 3350-3359). The native RNAase A, with the disulfide bridges intact, contained 19% helix and 38% beta-structure. Reduction of its disulfide bridges led to a decrease in the proportion of these structures to 9% for the alpha-helix and 17% for the beta-structure. The non-reduced RNAase A resisted unfolding in low concentrations of urea and guanidine hydrochloride. The beta-structure which remained after reduction appeared to be stable even in solutions of 6 M guanidine and 9 M urea. A considerable amount of the beta-structure in both the non-reduced and the reduced RNAase A remained unaffected by high concentrations of SDS.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

IR spectra of cytochrome c denatured with deuterated guanidine hydrochloride show increase in beta sheet

Attenuated total reflectance Fourier transform IR (ATR-FTIR) spectra are obtained for horse heart ferricytochrome c in solutions of 0-7M guanidine hydrochloride and deuterated guanidine hydrochloride. Substitutions of deuterium for hydrogen in both the denaturant and protein provide resolvable amide I spectra over a wide range of denaturant concentrations. Deuteration enhances the ability to measure the true protein IR spectrum in the amide I region in which the secondary structure can be deduced, because spectra in D(2)O are less prone to spectral distortion upon background denaturant subtraction than spectra in H(2)O. Other investigators studying equilibrium unfolded cytochrome c were limited to guanidine concentrations below 3.0M because of detector saturation. Detector saturation is avoided with the use of ATR-FTIR spectroscopy, allowing one to obtain protein spectra at high denaturant concentrations. Second derivative spectra of samples show reductions in alpha helix and increases in beta sheet at high denaturant concentrations, contrary to expectations of finding primarily a random coil secondary structure. Using this new technique, the protein was estimated to consist of 51% beta sheet and only 15% random coil in the presence of 6.6M deuterated guanidine hydrochloride.     

Purification and characterization of a molecular weight 100,000 coat protein from coated vesicles obtained from bovine brain

A protein designated as a 100-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis was purified from coated vesicles obtained from bovine brain, with uncoated vesicles as starting material. Two gel filtration steps, one involving 0.5 M tris(hydroxymethyl)aminomethane, pH 8.0, buffer, and the other 0.01 M tris(hydroxymethyl)aminomethane, pH 8.0, and 3 M urea buffer, were employed. The purified protein has a native molecular weight of 114,000 as determined by sedimentation equilibrium analysis. Circular dichroism data showed that the protein has 28% helical structure, 29% beta-structure, and 15% beta-turns, and the rest is random coil. Addition of the purified protein to clathrin results in the polymerization of clathrin to homogeneous size baskets of sedimentation velocity 150 S. A scan of the Coomassie Blue stained electrophoresis gels of the polymerized baskets shows that, for every clathrin trimer, there is approximately one 100-kDa protein molecule.     

岩豆凝集素的圆二色性与生物学活性关系的研究

岩豆凝集素（ＭＤＬ）的远紫外圆二色性谱（ＣＤ谱）显示２１６－２１７ｎｍ处的单一负峰。此时ＭＤＬ分子含有１６．２％的α螺旋,４６．３％的β折叠和３７．５％的无规卷曲。ｐＨ９．０时负峰红移至２２０ｎｍ,且在２１７－２２２ｎｍ处的峰值几乎相同;在２０－４０℃范围内,ＣＤ谱的变化甚微;６０℃时谱峰蓝移;在８０℃或１００℃时,２１２ｎｍ处出现一大负峰。１ｍｏｌ／Ｌ或２ｍｏｌ／Ｌ脲时,ＭＤＬ的ＣＤ谱已发生明显变化,二级结构单元也有变化,凝集兔红细胞的活性也随之减弱;随脲浓度的增加,ＭＤＬ的谱峰蓝移,最终在２１２ｎｍ处出现大负峰。当胍浓度为０．７５ｍｏｌ／Ｌ时,ＭＤＬ的ＣＤ谱即有明显变化和活性丧失;胍浓度继续增加,ＣＤ谱逐渐成为特征的无规卷曲的谱形。在ｐＨ９．０、温度超过８０℃、脲或胍浓度分别高于２ｍｏｌ／Ｌ和０．７５ｍｏｌ／Ｌ时,ＭＤＬ的ＣＤ谱发生显著变化的同时,其凝集兔红细胞的生物学活性全部丧失,分子的二级结构单元也发生很大改变。     

Ubiquitin-dependent proteolysis of native and alkylated bovine serum albumin: effects of protein structure and ATP concentration on selectivity

The susceptibility of bovine serum albumin to degradation by the ubiquitin-dependent system of proteolysis depends on the severity of the iodination conditions [Wilkinson, K.D., & Audhya, T.K. (1981) J. Biol. Chem. 256, 9235-9241]. To evaluate if other modifications of the protein changed its susceptibility to degradation, chemically modified derivatives of bovine serum albumin have been synthesized, characterized, and tested as substrates for the ubiquitin-dependent system. Serum albumin was reduced or reduced and alkylated with iodoacetic acid or iodoacetamide. Only the alkylated derivatives exhibit saturation kinetics. Both alkylated proteins competitively inhibit the degradation of the other. These substrates are useful for assay of the intact proteolysis system in crude extracts and in assays for other substrates using competitive alternate substrate inhibition. The physical properties of these proteins suggest that charge, denaturation, or aggregation is not correlated with the degradation rate of these proteins by this system. However, the selectivity of the ubiquitin-dependent proteolysis depends strongly on the ATP concentration. At saturating substrate concentrations, both alkylated substrates are degraded equally. At low ATP concentrations, there is a 2.4-fold difference in the degradation rates of the alkylated proteins. The results presented here indicate that the ubiquitin-dependent protein degradation system is selective and responsive to ATP concentrations and that not all abnormal proteins are equally preferred substrates. Thus, the system may be more selective than previously thought.     

Circular dichroism of platelet factor 4

The circular dichroism of platelet factor 4 was investigated and it was found to contain 15% alpha-helix, 25% beta-structure, and the rest of the molecule in unordered conformation. In the presence of heparin, no change in the circular dichroism was observed, suggesting no significant changes in the secondary structure of platelet factor 4 when heparin binds. The CD spectrum of platelet factor 4 was also investigated in the presence of increasing concentrations of guanidine hydrochloride. A two-state transition was observed with midpoints at 0.125 and 2.0 M guanidine hydrochloride. Based on gel filtration studies, the first unfolding transition was correlated with the dissociation of the tetrameric structure. This first unfolding domain was not observed in the presence of heparin, suggesting that heparin stabilizes the tetrameric structure. The second unfolding transition corresponds to the disruption of the overall secondary structure which is generally observed with most proteins. It is concluded that a relatively weak force of attraction holds the tetrameric structure of platelet factor 4 and the dissociation of the subunits is accompanied by loss of some helical secondary structure.     

Molten globule intermediates of human serum albumin in low concentration of urea

Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the alpha-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.     

The low-pH unfolded state of the C-terminal domain of the ribosomal protein L9 contains significant secondary structure in the absence of denaturant but is no more compact than the low-pH urea unfolded state

There is considerable interest in the properties of the unfolded states of proteins, particularly unfolded states which can be populated in the absence of high concentrations of denaturants. Interest in the unfolded state ensemble reflects the fact that it is the starting point for protein folding as well as the reference state for protein stability studies and can be the starting state for pathological aggregation. The unfolded state of the C-terminal domain (residues 58-149) of the ribosomal protein L9 (CTL9) can be populated in the absence of denaturant at low pH. CTL9 is a 92-residue globular alpha, beta protein. The low-pH unfolded state contains more secondary structure than the low-pH urea unfolded state, but it is not a molten globule. Backbone ( (1)H, (13)C, and (15)N) NMR assignments as well as side chain (13)C beta and (1)H beta assignments and (15)N R 2 values were obtained for the pH 2.0 unfolded form of CTL9 and for the urea unfolded state at pH 2.5. Analysis of the deviations of the chemical shifts from random coil values indicates that residues that comprise the two helices in the native state show a clear preference for adopting helical phi and psi angles in the pH 2.0 unfolded state. There is a less pronounced but nevertheless clear tendency for residues 107-124 to preferentially populate helical phi and psi values in the unfolded state. The urea unfolded state has no detectable tendency to populate any type of secondary structure even though it is as compact as the pH 2.0 unfolded state. Comparison of the two unfolded forms of CTL9 provides direct experimental evidence that states which differ significantly in their secondary structure can have identical hydrodynamic properties. This in turn demonstrates that global parameters such as R h or R g are very poor indicators of "random coil" behavior.     

Secondary structure of human interleukin-3

1. The secondary structure of human interleuken-3 in solution was determined by circular dichroism spectroscopy. 2. The results were then compared with empirical secondary structure predictions based on primary sequence structure of the protein. 3. The two approaches are in extremently close agreement showing the protein to have 40% alpha-helix, 12% total beta-structure and 48% random coil content.     

Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride

By analyzing the effect of urea and guanidine hydrochloride on the circular dichroism of many polypeptides and proteins, it is concluded that under conditions of high concentration of the perturbant and at low temperatures the resultant state approached is that of a local extended helix structure instead of a completely random coil. Intensification by urea and guanidine hydrochloride of the circular dichroism bands of poly-L -proline II leads to the proof that the mechanism of interaction of urea and guanidine hydrochloride with proteins is through hydrogen bonding to the backbone carbonyl group.     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.     

NATIVE AND REGENERATED BOVINE ALBUMIN : II. IMMUNOLOGICAL PROPERTIES

1. The effects of regeneration of whole bovine albumin on antigenic activity and serological specificity were determined by precipitin measurements on rabbit antisera to (1) native whole albumin, (2) albumin regenerated from 8 M urea, and (3) albumin regenerated from 8 M guanidine hydrochloride. 2. While numerically the mean antibody response to these three antigens was found to decrease in the order named, only the difference in antigenic activity between native and guanidine hydrochloride-regenerated albumin was statistically significant. Native, crystalline, carbohydrate-free albumin (crystalbumin) was considerably less antigenic than native whole bovine albumin, its activity being comparable to, if not less than, that observed for guanidine hydrochloride-regenerated whole albumin. 3. All four antigens were immunologically equivalent. 4. The antigenic activity of these proteins is discussed in terms of protein structure and carbohydrate content.     

Thermodynamic features of the chemical and thermal denaturations of human serum albumin

The unfolding process of human serum albumin between pH 5.4 and 9.9 was studied by chemical and thermal denaturations. The experimental results showed that there is no correlation between the stability of albumin at different pH values determined by both methods. The free energy change of unfolding versus concentration of guanidine showed a close dependence on the pH, suggesting that the variation of the electrical charge of albumin influences the final state of the unfolded form of the protein. Spectroscopic techniques, such as native fluorescence of the protein and circular dichroism, demonstrated that the unfolded state of the protein obtained from both methods possesses a different helical content. The solvophobic effect and the entropy of the chains have no influence on the final unfolding state when the protein is unfolded by thermal treatment, while, when the protein is unfolded by chemical denaturants, both effects depend on the medium pH. The results indicate that guanidine and urea interact with albumin by electrostatic forces, yielding a randomly coiled conformation in its unfolded state, while thermal denaturation produces a molten globule state and the aggregation of the protein; therefore, both methods yield different structurally unfolded states of the albumin.     

Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.     

An evaluation of chemical shift index-based secondary structure determination in proteins: Influence of random coil chemical shifts

Random coil chemical shifts are commonly used to detect protein secondary structural elements in chemical shift index (CSI) calculations. Though this technique is widely used and seems reliable for folded proteins, the choice of reference random coil chemical shift values can significantly alter the outcome of secondary structure estimation. In order to evaluate these effects, we present a comparison of secondary structure content calculated using CSI, based on five different reference random coil chemical shift value sets, to that derived from three-dimensional structures.Our results show that none of the reference random coil data sets chosen for evaluation fully reproduces the actual secondary structures. Among the reference values generally available to date, most tend to be good estimators only of helices. Based on our evaluation, we recommend the experimental values measured by Schwarzinger et al.(2000), and statistical values obtained by Lukin et al. (1997), as good estimators of both helical and sheet content.     

Contribution of disulfide bonds to stability of the folding intermediate of alpha-lactalbumin

The secondary structure formed in disulfide reduced alpha-lactalbumin is investigated by CD spectrum and is compared with that of the folding intermediate of the disulfide intact protein. The peptide backbone structure of the reduced protein depends strongly on salt concentration in contrast to that of the intermediate. It is close to a random coil in the absence of salt, but it is almost the same as that of the intermediate at a high concentration of salt. The secondary structures of both the proteins undergo broad unfolding transitions when temperature is raised or when urea is added. The secondary structure of the reduced protein is less stable against both heat and urea. These results show that the disulfide bonds are not a determinant of the secondary structure formed at an early stage of folding, and they stabilize the secondary structure of the folding intermediate.