Denaturation of a membrane transport protein by urea: The erythrocyte anion exchanger

Summary Chloride equilibrium exchange was measured in the presence of intracellular and extracellular urea, several different alkylureas and thiourea. Urea half-inhibited Cl exchange at about 2.5m, but the other, less polar analogs had significantly higher potencies; e.g., butylurea half-inhibited at about 60mm. Onset and reversal of inhibition occurred within less than 2 sec. The inhibition exhibited no obvious sigmoidal dependence on urea concentration, and at low concentrations dimethylurea was a noncompetitive inhibitor of Cl exchange. However, at higher concentrations the Dixon plots were curved upward and a Hill analysis of the dimethylurea data yielded a Hill coefficient of at least 1.5. When present on only one side of the membrane, the slowly penetrating thiourea inhibited Cl exchange with a higher potency from the outside of the cell. Cl/Br exchange was inhibited less under conditions of self-inhibition of anion exchange than in the absence of self-inhibition. These data indicate that the ureas inactivate the anion transporter by a reversible denaturation process, and that the function of the anion transport mechanism may be more sensitive to small perturbations of protein structure than are spectroscopically derived structural parameters.     

Rapid Denaturation Improves Chromosome Morphology and Permits Multiple Hybridizations During Fluorescence in Situ Hybridization

Denaturation of chromosomal DNA for fluorescence in situ hybridization (FISH) is an essential step in a procedure associated with a number of variables. In our experience, shorter denaturation time in 70% formamide/2 × SSC at 72 C provides sufficient denaturation, where the hydrogen bonds are broken between the purines and pyrimidines of the double helix. This shortened exposure improves retention of morphology of human chromosomes from lymphocytes, aminocytes, fibroblasts and bone marrow, and allows the same metaphases to be denatured repeatedly and rehybridized with different probes. This approach is useful in investigations where sample volume is limited.     

胶原蛋白水解肽磷酸化的研究

在三聚磷酸钠作用下,胶原活性肽能够发生非酶磷酸化,共价连接磷酸基团,用正交实验法优选磷酸化条件,反应温度对磷酸化水平影响最大,pH值影响次之,在优选的磷酸化条件下（pH9.0,80℃,反应3h),磷酸化水平约为每分子胶原能够共价连接上约2分子磷酸基团。     

Cold and Warm Denaturation of Proteins

We introduce a simplified protein model where the water degrees of freedom appear explicitly (although in an extremely simplified fashion). Using thismodel we are able to recover both the warm and the cold protein denaturation within a single framework, while addressing important issues about the structure of model proteins.     

Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.     

Statistical Mechanical Theory of DNA Denaturation

Thermodynamic quantities of a coupled sine-lattice chain, which is a simplified model of DNA (rotator model) are calculated for both in low and high temperature regions. In the high temperature region those quantities are expressed as a series expansion in terms of modified Bessel functions of an integer order. The results of numerical calculations are presented in connection with DNA denaturation (melting).     

羟基化修饰对大鼠胶原热稳定性的影响研究

采用差示扫描量热仪(DSC)研究羟脯氨酸(Hyp)含量对胶原热稳定性的影响。以不同周龄BN大鼠皮肤为原料制备了胶原,分析制备胶原中Hyp的含量;采用DSC测定不同Hyp含量胶原的临界变性温度及焓变;采用圆二色光谱(CD)检测提取胶原的二级结构。结果表明,提取胶原在41.3℃发生三螺旋解聚,CD光谱分析结果表明,当样品经临界变性温度处理后,部分三螺旋结构转化为无规则线圈结构。胶原变性过程中所需热量与羟脯氨酸含量呈正相关,实验建立了胶原热变性过程中焓变与Hyp含量的关系。该研究表明胶原中脯氨酸羟基化修饰是影响胶原热稳定性的关键因素。     

Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation

FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers.     

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the protein decorated micelle structure or the necklace structure.     

大肠杆菌异源重组蛋白质的变性与复性

描述了大肠杆菌异源重组蛋白质的形成、制备、变性和复性,综述了国内外变性、复性的有效方法。     

抗鱼肉蛋白冷冻变性机理的研究进展

简述了鱼肉蛋白冷冻变性机理的研究现状,综述了糖类、盐类、乳蛋白、不同水解物等添加物的抗冷冻变性机理,介绍了鱼肉蛋白冷冻变性的评价指标及其测定方法,展望了抗鱼肉蛋白冷冻变性的新途径及应用前景。论文内容对于深入研究抗肉类蛋白冷冻变性具有较大的参考价值。     

火菇素的溶液二级结构与变性动力学

为了弄清抗肿瘤活性物质火菇素蛋白的二级结构及其活性在保存时自然衰减的规律 ,为临床应用提供依据 ,测定了火菇素的圆二色性并用蛋白质二级结构解析程序分析了火菇素的溶液二级结构 ,研究了火菇素的变性动力学 .火菇素的远紫外圆二色性的研究表明 ,其水溶液在 2 0 8nm处表现为大负峰 ,最大平均残基摩尔椭圆度 [θ]2 0 8=- 6574deg·cm2·dmol-1,在 2 2 3nm处为肩 .经二级结构解析程序计算分析 ,火菇素的二级结构组成为 :α螺旋 1 5.2 % ,平行 β折叠片和 β转角6.1 % ,反平行 β折叠片 32 .7% ,无规卷曲和 γ转角 2 3.4% ,其中二硫键和芳香氨基酸对火菇素圆二色性的贡献占 2 2 .6% .热变性几乎使所有的二级结构都遭到破坏 ,转化为无规卷曲 .利用已建立的火菇素免疫单向琼脂扩散定量检测技术 ,对在 4℃下保存的火菇素进行了长期跟踪检测 ,结果表明 ,在保存过程中火菇素的活性逐步降低 ,同时其二级结构也被破坏 .根据实验结果 ,建立了火菇素变性的一级动力学方程模型 :ct=coe-t/τ,该模型方程能很好地拟合实验结果 .根据模型方程计算的火菇素的寿命为 370 d,半衰期为 2 56d.说明火菇素这种很有应用前景的抗肿瘤药物比较容易长期保存 .     

Mechanism of the interactions of aliphatic alcohols with bovine serum albumin in the ternary systems—1H n.m.r. study: 1. Aqueous solutions BSA-alcohols-urea

The molecular mechanism of the interaction of aliphatic alcohols (A) with bovine serum albumin (BSA) protein was studied in aqueous solutions at increasing concentrations (0–8 m) of urea (U). ¹H n.m.r. spectra of alcohols were monitored in D₂O in the control binary systems (A—U) and (A—BSA), and in the ternary systems (A—U—BSA) at pH 7.0. Marked and selective broadening of the n.m.r. lines of alcohols in the system (A—BSA) was reduced upon addition of urea, indicating that alcohols are poorly bound by urea-denaturated BSA. The reduction in the ability to associate with BSA depends on chain position of the alcohol molecule and is much higher for α-methylenes (next to ?OH) than for other proton groups. Besides this reduction seems to be a two-step phenomenon dependent upon urea concentration. The results obtained can be explained by competition in formation by the peptide linkages of a protein of the hydrogen bonds with ?OH group of alcohols or fragments of urea molecules.     

Appearance of Nitrite Reducing Activity of Cytochrome c upon Heat Denaturation

The appearance of NO₂ ^? reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100°C for 30 min, had NO₂ ^? reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO₂ ^? reduction to NH₄ ⁺ via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.     

Approaches for increasing the solution stability of proteins

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.     

重组人GM—CSF／MCAF融合蛋白的变性,复性及纯化研究

人粒细胞巨噬细胞集落刺激因子(GM-CSF)和单核细胞趋化激活因子(MCAF)融合蛋白在大肠杆菌中高效表达后,表达产物以包涵体形式存在。包涵体经分离和洗涤后,探索了rhGM-CSF/MCAF变性和复性的合适条件。复性后的样品经Sephadex G-75凝胶过滤和CM-Sepharose FF离子交换两步层析,得到了具有生物学活性的SDS-PAGE纯的rhGM-CSF/MCAF。Western blot检测表明,纯化的rhGM-CSF/MCAF能分别与GM-CSF和MCAF抗体发生特异反应。     

Association-dissociation of glycinin in urea,guanidine hydrochloride and sodium dodecyl sulphate solutions

The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a ‘2S’ protein through the intermediary 7S and 4S proteins. Howeαer, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S_20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the aboαe reagents but to different extents.     

Denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions

The denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation of phaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases.     

A Model for the Unusual Kinetics of Thermal Denaturation of Rubredoxin

The thermal denaturation of the simple, redox-active iron protein rubredoxin is characterized by a slow, irreversible decay of the characteristic red color of the iron center at elevated temperatures in the presence of oxygen at pH 7.8. The denaturation rate is essentially constant and the time period for complete bleaching is nearly independent of protein concentration. These two characteristics of the kinetics can be fit by a simple self-catalyzed kinetics model consisting of the combination of a first-order decay and catalysis by some product of that decay, i.e., dP/dt=k ₁[A]+(k ₂[P][A])/(K _m+[A]), where A is native rubredoxin, P, is unspecified product, k ₁ is a first-order rate constant, and k ₂ and K _m are the catalytic constants. In order for the second term to be of this simple form over the full course of a decay, the model must include the condition that the reaction is effectively irreversible. This model has properties which suggest other biological roles in regulation (changes in k ₁ or k ₂ can dramatically modulate the kinetics), in timing (titer-independent fixed reaction time), and in self-activation reactions. At one extreme (k₁ k₂) the kinetics becomes exponential, but at the other extreme (k₂ k₁) they show a dramatic and rapid terminal increase after a lag period. Some obvious possible roles in the kinetics of programmed cell death, prion disease, and protease autoactivation are discussed.     

A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.