Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule

The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.     

Peptide models of local and long-range interactions in the molten globule state of human alpha-lactalbumin

alpha-Lactalbumin, a small calcium-binding protein, forms an equilibrium molten globule state under a variety of conditions. A set of four peptides designed to probe the role of local interactions and the role of potential long-range interactions in stabilizing the molten globule of alpha-lactalbumin has been prepared. The first peptide consists of residues 20 through 36 of human alpha-lactalbumin and includes the entire B-helix. This peptide is unstructured in solution as judged by CD. The second peptide is derived from residues 101 through 120 and contains both the D and 310 helices. When this peptide is crosslinked via the native 28 to 111 disulfide to the B-helix peptide, a dramatic increase in helicity is observed. The crosslinked peptide is monomeric, as judged by analytical ultracentrifugation. The peptide binds 1-anilinonaphthalene-8-sulphonate (ANS) and the fluorescence emission maximum of the construct is consistent with partial solvent exposure of the tryptophan residues. The peptide corresponding to residues 101 to 120 adopts significant non-random structure in aqueous solution at low pH. Two hydrophobic clusters, one involving residues 101 through 104 and the other residues 115 through 119 have been identified and characterized by NMR. The hydrophobic cluster formed by residues 101 through 104 is still present in a smaller peptide containing only residues 101 to 111 of alpha-lactalbumin. The cluster also persists in 6 M urea. A non-native, pH-dependent interaction between the Y103 and H107 side-chains that was previously identified in the acid-denatured molten globule state was examined. This interaction was found to be more prevalent at low pH and may therefore be an example of a local interaction that stabilizes preferentially the acid-induced molten globule state.     

The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin

The chaperone-like alpha-crystallin prevents aggregation of several proteins by interacting with their non-native states. Alpha-Lactalbumin adopts different non-native states under different experimental conditions. We have investigated the interaction of alpha-crystallin with three non-identical non-native states, using fluorescence, circular dichroism, and gel filtration chromatography. The compact molten globule state of apo-alpha-lactalbumin in tris buffer does not interact with alpha-crystallin. The expanded, flexible molten globule-like state of reduced apo-alpha-lactalbumin (formed at pH 7.2) also does not interact with alpha-crystallin. Only the aggregation-prone non-native state of reduced apo-alpha-lactalbumin formed at pH 6.0 interacts with alpha-crystallin to form a stable complex. The alpha-crystallin bound reduced apo-alpha-lactalbumin exhibits properties similar to those of a molten globule. Our results show that alpha-crystallin interacts only with the aggregation prone molten globule state of reduced apo-alpha-lactalbumin but not with the other non-aggregating molten globule states of the protein.     

Hydrophobic sequence minimization of the alpha-lactalbumin molten globule

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the alpha-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native alpha-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.     

The herpes simplex virus triplex protein, VP23, exists as a molten globule

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.     

The native-like tertiary fold in molten globule alpha-lactalbumin appears to be controlled by a continuous phase transition

On account of its ability to discriminate between secondary, loop and sidegroup structure and its special sensitivity to conformational mobility, vibrational Raman optical activity (ROA) has provided new insights into the complexity of order within the molten globule state from measurements on alpha-lactalbumin at pH 2.0 over the temperature range 2 to 45 degrees C. Thus while much of the secondary structure present in the native protein persists with only a small gradual decrease with increasing temperature, the tertiary backbone fold changes dramatically, being almost complete and native-like at 2 degrees C and almost completely disordered at 35 degrees C. The change of the tertiary fold with temperature is cooperative but has no latent heat, and so has the approximate characteristics of a continuous phase transition, being of the order-disorder type since it involves the interconversion of rigid, locally-ordered loop structure with disordered mobile backbone structure. This has implications for protein folding because the long-range correlations that exist in the critical region of a continuous (but not in a first-order) phase transition could resolve, in principle, the problem of how the protein finds its native-like folding pattern at the molten globule stage.     

Interaction of copper sulfides with copper oxides in the molten state

Reactions of Cu₂S with Cu₂O, CuS with Cu₂O and CuS with CuO in the molten state were examined in the presence of one atmosphere of argon at 1200°C. A rate law of the form,r _SO2 =kN_SN_O was applicable for each reaction system studied. Comparison of the rate constants for the systems, under conditions of similar initial mole fraction of sulfur to mole fraction of oxygen ratios, showed that Cu₂O was much more reactive than CuO in its reaction with copper sulfides. These results are incorporated in a mechanism in which Cu₂O reacts with the sulfide in the rate determining step. Experiments carried out in the presence of oxygen indicated the importance of a CuO-Cu₂O equilibrium in the overall reaction mechanism.     

Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten globule, and unfolded states

The volume change for the transition from the native state of horse heart apomyoglobin to a pressure-induced intermediate with fluorescence properties similar to those of the well-established molten globule or I form was measured to be -70 ml/mol. Complete unfolding of the protein by pressure at pH 4.2 revealed an upper limit for the unfolding of the intermediate of -61 ml/mol. At 0.3 M guanidine hydrochloride, the entire transition from native to molten globule to unfolded state was observed in the available pressure range below 2.5 kbar. The volume change for the N-->I transition is relatively large and does not correlate well with the changes in relative hydration for these transitions derived from measurements of the changes in heat capacity, consistent with the previously observed lack of correlation between the m-value for denaturant-induced transitions and the measured volume change of unfolding for cooperativity mutants of staphylococcal nuclease (Frye et al. 1996. Biochemistry. 35:10234-10239). Our results support the hypothesis that the volume change associated with the hydration of protein surface upon unfolding may involve both positive and negative underlying contributions that effectively cancel, and that the measured volume changes for protein structural transitions arise from another source, perhaps the elimination of void volume due to packing defects in the structured chains.     

Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.     

Free CaO stabilisation by mixing of BF slag and BOF slag in molten state

Reducing free CaO (f-CaO) in basic oxygen furnace (BOF) slag effectively and efficiently is the prerequisite to achieve the goal of BOF slag's volume stability. In this work, Taguchi experimental design method was used to explore the influencing factors effect on stabilising f-CaO by mixing BOF slag with blast furnace (BF) slag and probe into the relevant mechanism. The results show that, in the setting conditions of this work, the most influential factor on the stabilisation ratio of f-CaO is mass ratio between BF slag and BOF slag, and then mixing temperature and duration time as a queue. The reasonable conditions are obtained as follows: mass ratio of BF slag to BOF slag is 6:1, temperature of mixing is 1783?K and duration time for mixing is 20?min. The f-CaO stabilised mechanism on BF slag and BOF slag molten mixing was regarded as mutual coupling effects of stabilisation reactions and dilution.     

Sequential unfolding of novelty and pleasantness appraisals of odors: Evidence from facial electromyography and autonomic reactions.

We investigated the effects of odors on appraisal processes and consequent emotional responses. The main goal was to test whether an odor is detected as novel or familiar before it is evaluated as pleasant or unpleasant. Participants performed a recognition task in which they were presented with pairs of unpleasant or pleasant odors (sample and target odors). Within a pair, the sample and target were either identical or different to assess participants’ novelty detection; unpleasant and pleasant target odors were contrasted to examine participants’ appraisal of intrinsic pleasantness. We measured facial expressions using electromyography and physiological reactions using electrocardiogram and electrodermal activity in response to odors. The earliest effects on facial muscles and heart rate occurred in response to novelty detection. Later effects on facial muscles and heart rate were related to pleasantness evaluation. This study is the first to demonstrate the existence of a sequence of appraisal checks for odors eliciting emotional reaction. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Conformational transitions provoked by organic solvents in beta-lactoglobulin: can a molten globule like intermediate be induced by the decrease in dielectric constant?

The macroscopic arrangement of the termination of the thoracic duct (TD) was examined in detail in Japanese cadavers, and the distribution of various types of collagen, tenascin, laminin, and fibronectin in the framework of the wall of the thoracic duct termination was demonstrated. We identified several branching patterns and pathways of the TD (i.e., those terminating 1) at the venous angle (Type A); 2) at the end of the internal jugular vein (Type B); 3) at the the external jugular vein (Type C); or 4) in a complex with various branches (Type D). The TD often divided into two or three trunks before or after passing beneath the left brachiocephalic vein. Compared with the main trunk of the TD, fewer muscle fibers, elastic fibers, specific compounds of the extracellular matrices, and valvular connective tissues were found near the termination of the TD and the valves of the TD and veins. Smooth muscle cells were arranged irregularly in each region of the TD.     

基于不同保温措施下的铁水包热状态模拟分析

在铁钢界面现有模式下的铁水运输过程中,由于铁水包运行周期及保温效果不够理想,导致在高炉接铁时铁包耐材温度低,热状态差,使得铁水在铁水包内的热量损失较大.减小铁水温降能有效防止铁水包结壳结瘤,降低离线烘烤频率,间接提高铁水包周转率;同时在转炉冶炼过程中,低温铁水将严重影响废钢的加入量和吹氧等操作.由此可见,铁水温度控制是钢铁企业节能降耗和高效有序生产的关键因素之一.为了减小铁水温降,本文建立了多种不同保温措施情况下的铁水包传热模型,通过fluent软件对各模型在不同空包时间情况下的温度场进行数值计算,分析不同保温措施及空包时间下热状态对铁水温降的影响规律.分析结果表明：无保温措施的情况下空包时间由5 h缩短至3 h能降低下一周期铁水温降2.2 K·h^-1;空包阶段最合理的保温措施为增设6 mm左右绝热层并加包盖,能提高工作层平均温度约155 K,在空包3~5 h内能减小铁水温降3.4~3.7 K·h^-1.该结论为铁水包空包阶段采取合理保温措施及不同保温情况下空包运行时间控制提供了理论指导.     

The globule leucocyte in bovine schistosomiasis

Globule leucocytes were detected in forestomachs, abomasum, duodenum, ileum, large intestine, lung, liver, bladder and kidney in cattle infected with Schistosoma mattheei. In animals examined seven and eight weeks after infection they were found only in the lungs. They were present in the alimentary tract from 18 weeks onwards. They were most numerous and most widely distributed in animals subjected to repeated, heavy infection. There was evidence that they were associated with the immune response of the host and that they were derived from mast cells.     

Chemical state of fluorine in fluoroaluminosilicate slags in glassy and molten states from perspective of electronic polarisability

Abstract

The electronic polarisability of F α _F has been determined for fluoroaluminosilicate slags from density and refractive index data measured to investigate the chemical state of F in the slags and the effect of CaF₂ additions on depolymerisation of the aluminosilicate network. To simulate commercial mould fluxes, synthetic slags with compositions of xCaF₂–5Na₂O–6Al₂O₃–(89–x)CaO.SiO₂ were prepared as samples, where x ranged between 5 and 12·5 mol.-%. Densities and refractive indices of the slags were measured in glassy and molten states; for the melts, the sessile drop method and ellipsometry were applied to density and refractive index measurements respectively. Values of α _F determined for the slags are on the whole in good agreement with those for CaF₂, irrespective of temperature, which suggests that F ions in the slags are in ionic bonding of Ca–F with a possibility of a Na–F bond. However, only a glassy slag with a lower concentration of CaF₂ exhibits a slightly smaller α _F than CaF₂, which would be a sign of Al–F and/or Si–F bonds being produced. From the value of α _F, F ions are likely to terminate silicate anions by forming Ca–F bonds in the slags and depolymerisation of the network by this termination would be responsible for lowering the viscosity of molten slags with CaF₂ additions.