Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae

The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI+] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI+] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are “cured” of [PSI+] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI+] determinant is not derived from the direct dissolution of self-replicating [PSI+] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI+] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI+] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.

SecB binds only to a late native-like intermediate in the folding pathway of barstar and not to the unfolded state

SecB is a cytosolic, tetrameric chaperone of Escherichia coli which maintains precursor proteins in a translocation competent state. We have investigated the effect of SecB on the refolding kinetics of the small protein barstar in I M guanidine hydrochloride at pH 7.0 and 25 degrees C using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to a late near-native intermediate along the folding pathway. For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models.

The effect of pH on the unfolding pathway and stability of ribosome-inactivating protein abrin-II

The effect of pH on the unfolding pathway acid the stability of the toxic protein abrin-II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1-anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV-circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin-II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin-II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin-II A-subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.

Studies on nucleotidases in plants : Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect. The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The Km (0.2 mImage ) and V (3.2) of the native enzyme increased on renaturation to 1.8 mImage and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn2+.

Studies on nucleotidases in plants - Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect. The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The Km (0.2 m ) and V (3.2) of the native enzyme increased on renaturation to 1.8 m and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn2+.

The denaturation of β-lactoglobulin-A at pH 2

The denaturation of β-lactoglobulin-A by heat and guanidine hydrochloride at pH 2 has been investigated. The effect of ethylene glycol on the thermal denaturation at this pH has also been studied. The conditions of the experiments have been chosen so as to eliminate complications arising out of disulfide interchange, changes in the degree of association of the protein during denaturation, and intermolecular aggregation. The physical parameters characterizing the denatured states of the protein which are produced by heat and guanidine hydrochloride have been determined. The thermodynamic parameters for these transitions have been estimated using a two-state hypothesis in each case. Both the physical and thermodynamic parameters indicate that the heat-denatured state of β-lactoglobulin-A retains about 15-20% of residual structure which is destroyed on adding guanidine hydrochloride.

Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

The Folding Mechanism of Barstar: Evidence for Multiple Pathways and Multiple Intermediates

The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25 degrees C has been characterized using rapid mixing techniques. Earlier studies had established the validity of the three-state U-S reversible arrow U-F reversible arrow N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where U-S and U-F are the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, I-S1 and I-S2 as well as a late native-like intermediate, I-N, are present on the folding pathways of U-S, and an early intermediate I-F1 on the folding pathway of U-F, when bars tar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of I-N, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, I-N is not fully populated on the U-S --> I-S1 --> I-N --> N pathway because the rate of its formation is so slow that the U-S reversible arrow U-F reversible arrow N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the U-S --> I-S1 --> I-N transition is so fast that I-N is fully populated. In 0.6 M GdnHCl, I-N appears not to be fully populated because an alternative folding pathway, U-S --> I-S2 --> N, becomes available for the folding of U-S, in addition to the U-S --> I-S1 --> I-N --> N pathway Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, I-M1 and I-M2, that form within 2 ms on the U-S --> I-M1 --> I-S1 --> I-N --> N and U-S --> I-M2 --> I-S2 --> N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of U-F.

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large [Delta]Cp's

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274–329 K. At 298 K, values of [Delta]G°, [Delta]Cp, and Cm were 3.1 ± 0.2 kcal mol−1, 5.9 ± 0.8 kcal mol−1 K−1 (15.9 cal (mol-residue)−1 K−1), and 0.8 M, respectively, at pH 3.0 and 14.5 ± 0.4 kcal mol−1, 8.3 ± 0.7 kcal mol−1 K−1 (22.4 kcal (mol-residue)−1 K−1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of [Delta]G° and [Delta]Cp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of [Delta]Cp per mol-residue for the molten globule is comparable to corresponding values of [Delta]Cp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of [Delta]Cp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.

理化因子诱导的CP43和CP47结构与功能变化规律的研究

CP43和CP47是PSII中位于类囊体膜上的两种内周天线色素蛋白复合体，它们都是由六个跨膜的α-螺旋和五个膜外环组成。CP43和CP47的主要功能是把光系统II(PSII)外周天线色素蛋白复合体(LHCII)吸收的能量传给反应中心(RC)，从而引起光化学反应。因此，研究CP43和CP47的结构与功能对于揭示植物光合作用高效吸能、传能的分子机理具有重要意义。由于CP43和CP47的分离纯化比较困难，所以相对于其它的光合膜蛋白来说，人们对CP43和CP47的研究比较少。在本文中，我们在分离、纯化CP43和CP47的基础上，采用多种光谱学和波谱学技术对CP43和CP47在GuHCl和高温作用下的变性过程及其结构与功能的变化规律进行了比较深入的研究，获得了如下结果： 1. CP43和CP47膜外区的结构特点及盐酸胍(GuHCl)引起的变性研究 我们用荧光光谱、园二色(CD)光谱研究了GuHCl引起CP43和CP47的变性过程及其膜外区的结构特点。研究发现：CP43和CP47的膜外区具有一定的有序结构，而不是一种没有规则的伸展状态;和CP43相比，CP47的三级结构及Chl a的微环境对GuHCl更敏感。在GuHCl作用下，从β-Car到Chl a的能量传递变化和三级结构的变化密切相关，而与二级结构变化的相关性则较小;和大多数水溶性蛋白不一样，CP43和CP47对GuHCl变性有一定的抵抗力，而且其变性过程不表现为二态过程，这些都是膜蛋白的特点。 2 CP43和CP47中与芳香族氨基酸有关的能量传递研究 我们用吸收光谱、荧光光谱并参照PSII的3.5 Å的晶体结构分析结果研究了CP43和CP47中与芳香族氨基酸有关的能量传递。发现：和水溶性蛋白不一样，CP43和CP47中的酪氨酸(Tyrs)并不能有效的把其能量传给色氨酸(Trps);CP43和CP47中的芳香族氨基酸能通过Föster机制和Dexter机制把其能量传给Chl a，并且CP47中的传递效率要大于CP43;在CP47中Föster机制是芳香族氨基酸和Chl a之间能量传递的主要方式，而在CP43中Dexter机制则是主要方式。这些结果也暗示了，太阳光中的紫外辐射对植物来说除了其伤害作用以外也有一定的益处。 3 GuHCl诱导CP43和CP47变性的太赫兹（THz）光谱研究 THz时域光谱技术(THz-TDS)是研究分子构型状态的一个新工具。近年来，已被应用于物理或化学分子的研究中。我们首次把这个技术应用到光合膜蛋白CP43和CP47的GuHCl变性研究上。研究发现，在小于1.5 THz时，THz吸收光谱强度随着频率的增加而增加可以看作是蛋白质变性的标志。在GuHCl作用下频域光谱中出现的1.8 THz峰应来源于Chl a和GuHCl之间的相互作用。实验结果表明，THz光谱是区分蛋白分子的不同构型状态以及监测蛋白变性过程的有力工具。 4 CP43热变性的傅立叶变换红外光谱和THz光谱研究 我们用傅立叶变换红外光谱技术(FT-IR)、SDS聚丙稀凝胶电泳(SDS-PAGE)和THz光谱技术对CP43的热变性过程进行了研究。结果表明，在高温处理下，CP43的二级结构发生了变化，且其跃变点发生在59℃。随着温度的逐渐升高，CP43先发生凝集，接着又发生降解;CP43的低频振动模随着温度的升高和分子量的减小也发生变化。我们还证实THz光谱技术在监测膜蛋白的热变性时既有它的优越性，也存在一些不足之处。这些结果为THz-TDS技术在生物样品上的应用提供了基本的资料，并完善了相关的理论。

A universal measure of chaotropicity and kosmotropicity

Diverse parameters, including chaotropicity, can limit the function of cellular systems and thereby determine the extent of Earth's biosphere. Whereas parameters such as temperature, hydrophobicity, pressure, pH, Hofmeister effects, and water activity can be quantified via standard scales of measurement, the chao-/kosmotropic activities of environmentally ubiquitous substances have no widely accepted, universal scale. We developed an assay to determine and quantify chao-/kosmotropicity for 97 chemically diverse substances that can be universally applied to all solutes. This scale is numerically continuous for the solutes assayed (from +361kJkg^-1mol^-1 for chaotropes to -659kJkg^-1mol^-1 for kosmotropes) but there are key points that delineate (i) chaotropic from kosmotropic substances (i.e. chaotropes =+4; kosmotropes =-4kJkg^-1mol^-1); and (ii) chaotropic solutes that are readily water-soluble (log P<1.9) from hydrophobic substances that exert their chaotropic activity, by proxy, from within the hydrophobic domains of macromolecular systems (log P>1.9). Examples of chao-/kosmotropicity values are, for chaotropes: phenol +143, CaCl₂ +92.2, MgCl₂ +54.0, butanol +37.4, guanidine hydrochloride +31.9, urea +16.6, glycerol [>6.5M] +6.34, ethanol +5.93, fructose +4.56; for kosmotropes: proline -5.76, sucrose -6.92, dimethylsulphoxide (DMSO) -9.72, mannitol -6.69, trehalose -10.6, NaCl -11.0, glycine -14.2, ammonium sulfate -66.9, polyethylene glycol- (PEG-)1000 -126; and for relatively neutral solutes: methanol, +3.12, ethylene glycol +1.66, glucose +1.19, glycerol [<5M] +1.06, maltose -1.43 (kJkg^-1mol^-1). The data obtained correlate with solute interactions with, and structure-function changes in, enzymes and membranes. We discuss the implications for diverse fields including microbial ecology, biotechnology and astrobiology.

Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228–259, 2015a, doi:10.​1016/​j.​copbio.​2015.​02.​010; Ball and Hallsworth in Phys Chem Chem Phys 17:8297–8305, 2015, doi:10.​1039/​C4CP04564E; Cray et al. in Environ Microbiol 15:287–296, 2013a, doi:10.​1111/​1462-2920.​12018). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight−1. Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0–85.7 kJ kg−1). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as ‘halophile’ and ‘polyextremophile’; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.