Protein folding forces

We investigate the average inter-residue folding forces derived from mutational data of the 15 proteins: barstar, barnase, chymotrypsin inhibitor 2 (CI2), Src SH3 domain, spectrin R16 domain, Arc repressor, apo-azurin, cold shock protein B (cspB), C-terminal domain of ribosomal protein L9 (CTL9), FKBP12, α-lactalbumin, colicin E7 immunity protein 7 (IM7), colicin E9 immunity protein 9 (IM9), spectrin R17 domain, and ubiquitin. The residue-specific contributions to folding in most of the 15 protein molecules are highly non-uniformly distributed and are typically about 1 piconewton (pN) per interaction. The strongest folding forces often occur in some of the helices and strands of folding nuclei which suggests that folding nucleation−condensation is partially directed by formation of some secondary structure interactions. The correlation of the energy changes of mutants with inter-residue contact maps of the protein molecules provides a higher resolution than assigning the mutant data to certain positions in the polypeptide strand alone. In contrast to previous Φ-value analysis, we now can partially resolve folding motions. Compaction of at least one α-helix along its axis mediated by internal hydrogen bonds and stabilized by diffuse tertiary structure interactions appears to be one important molecular event during early folding in barstar, CI2, spectrin R16 domain, Arc repressor, α-lactalbumin, IM7, IM9, and spectrin R17 domain. A lateral movement of at least two strands neighbored in sequence towards each other appears to be involved in early folding of the SH3 domain, cspB, CTL9, and FKBP12.     

Folding of the alphaII-spectrin SH3 domain under physiological salt conditions

The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of αII-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the αII-spectrin SH3 domain.     

Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects

Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed α-β protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 °C. Plots of the log of the observed first-order rate constant versus denaturant concentration, “chevron plots,” displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford β parameter, β_T, shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of ΔG^‡versus ΔG°, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.     

Effect of heat treatment on lactoperoxidase activity in caprine milk

Using isothermal conditions, inactivation of lactoperoxidase (LPO) in caprine milk was studied in a temperature range of 69–73 °C. In order to evaluate the effect of temperature on the reaction rate, the Arrhenius and thermal death time models were used for data analysis. Thermal inactivation of LPO can be accurately described by a first-order kinetic model, as indicated by the relationships obtained by plotting the retention values as a function of treatment time on a semi-logarithmic scale and confirmed by the high R²-values obtained. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The corresponding Z- and E_a-values calculated from the slope of the semi-logarithmic plots of D and k as a function of temperature were 9.45 °C and 225.98 kJ/mol, respectively.     

An approach to branched-chain amino sugars,particularly derivatives of l-vancosamine (3-amino-2,3,6-trideoxy-3-C-methyl-l-lyxo-hexose) and its d enantiomer,via the cyanohydrin route

Methyl 4,6-O-benzylidene-2-deoxy-α-d-erythro-hexopyranosid-3-ulose reacted with potassium cyanide under equilibrating conditions to give, initially, methyl 4,6-O-benzylidene-3-C-cyano-2-deoxy-α-d-ribo-hexopyranoside (7), which, because it reverted slowly to the thermodynamically stable d-arabino isomer, could be crystallised directly from the reaction mixture. The mesylate derived from the kinetic product 7 could be converted by published procedures into methyl 3-acetamido-2,3,6-trideoxy-3-C-methyl-α-d-arabino-hexopyranoside, which was transformed into methyl N-acetyl-α-d-vancosaminide on inversion of the configuration at C-4. A related approach employing methyl 2,6-dideoxy-4-O-methoxymethyl-α-l-erythro-hexopyranosid-3-ulose gave the kinetic cyanohydrin and thence, via the spiro-aziridine 27, methyl 3-acetamido-2,3,6-trideoxy-3-C-methyl-α-l-arabino-hexopyranoside, a known precursor of methyl N-acetyl-α-l-vancosaminide.     

Linkage analysis in the presence of errors II: marker-locus genotyping errors modeled with hypercomplex recombination fractions

It is well known that genotyping errors lead to loss of power in gene-mapping studies and underestimation of the strength of correlations between trait- and marker-locus genotypes. In two-point linkage analysis, these errors can be absorbed in an inflated recombination-fraction estimate, leaving the test statistic quite robust. In multipoint analysis, however, genotyping errors can easily result in false exclusion of the true location of a disease-predisposing gene. In a companion article, we described a "complex-valued" extension of the recombination fraction to accommodate errors in the assignment of trait-locus genotypes, leading to a multipoint LOD score with the same robustness to errors in trait-locus genotypes that is seen with the conventional two-point LOD score. Here, a further extension of this model to "hypercomplex-valued" recombination fractions (hereafter referred to as "hypercomplex recombination fractions") is presented, to handle random and systematic sources of marker-locus genotyping errors. This leads to a multipoint method (either "model-based" or "model-free") with the same robustness to marker-locus genotyping errors that is seen with conventional two-point analysis but with the advantage that multiple marker loci can be used jointly to increase meiotic informativeness. The cost of this increased robustness is a decrease in fine-scale resolution of the estimated map location of the trait locus, in comparison with traditional multipoint analysis. This probability model further leads to algorithms for the estimation of the lower bounds for the error rates for genomewide and locus-specific genotyping, based on the null-hypothesis distribution of the LOD-score statistic in the presence of such errors. It is argued that those genome scans in which the LOD score is 0 for >50% of the genome are likely to be characterized by high rates of genotyping errors in general.     

Origin of unusual phi-values in protein folding: evidence against specific nucleation sites

phi(f)-value analysis is one of the most common methods to characterize the structure of protein folding transition states. It compares the effects of mutations on the folding kinetics with the respective effects on equilibrium stability. The interpretation of the results usually focuses on a few unusual phi(f)-values, which are either particularly high or which are larger than 1 or smaller than 0. These mutations are believed to affect the most important regions for the folding process. A major uncertainty in experimental phi(f)-values is introduced by the commonly used analysis of only a single mutant at various positions in a protein (two-point analysis). To test the reliability of two-point phi(f)-values we used reference data from three positions in two different proteins at which multiple mutations have been introduced. The results show that two-point phi(f)-values are highly inaccurate if the difference in stability between two variants is less than 7 kJ/mol, corresponding to a 20-fold difference in equilibrium constant. Comparison with reported phi(f)-values for 11 proteins shows that most unusual phi(f)-values are observed in mutants which show changes in protein stability that are too small to allow a reliable analysis. The results argue against specific nucleation sites in protein folding and give a picture of transition states as distorted native states for the major part of a protein or for large substructures.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythorocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin_kinase according and site-specificity criteria. The soluble enzyme shows an M_r of about 30 000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32 000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that has been phosphorylated with [γ-³²P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21 500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with ³²P_i. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Analysis of permethylated hexopyranosyl-2-acetamido-2-deoxyhexitols by g.l.c.-m.s.

The major product obtained on acetonation of d-mannose with a 2-molar excess of isopropenyl methyl (or ethyl) ether is 4,6-O-isopropylidene-α-d-mannopyranose (3a), the product of kinetic acetonation: a larger excess of the reagent leads, to the 2,3:4,6-diisopropylidene acetal (6). The course of the reaction and side-products formed were examined in detail. The 1,2,3-triacetate of 3a was deacetonated to give α-d-mannopyranose 1,2,3-triacetate; similar reactions were performed on the β anomers. The 1-acetate of the diacetal 6 could be selectively deacetonated to give 1-O-acetyl-2,3-O-isopropylidene-α-d-mannopyranose. The reactions provide access to protected derivatives of d-mannose, and partially acylated derivatives, having modes of substitution different from those obtainable by classical acetonation procedures conducted under conditions of thermodynamic control.     

Roles of physical interactions in determining protein-folding mechanisms: molecular simulation of protein G and alpha spectrin SH3

Protein-folding mechanisms of two small globular proteins, IgG binding domain of protein G and alpha spectrin SH3 domain are investigated via Brownian dynamics simulations with a model made of coarse-grained physical energy functions responsible for sequence-specific interactions and weak Gō-like energies. The folding pathways of alpha spectrin SH3 are known to be mainly controlled by the native topology, while protein G folding is anticipated to be more sensitive to the sequence-specific effects than native topology. We found in the folding of protein G that the C terminal beta hairpin is formed earlier and is rigid, once ordered, in the presence of an intact C terminal turn. The alpha helix is found to exhibit repeated partial formations/deformations during folding and to be stabilized via the tertiary contact with preformed beta sheets. This predicted scenario is fully consistent with experimental phi value data. Moreover, we found that the folding route is critically affected when the hydrophobic interaction is excluded from physical energy terms, suggesting that the hydrophobicity critically contributes to the folding propensity of protein G. For the folding of alpha spectrin SH3, we found that the distal beta hairpin and diverging turn are parts formed early, fully in harmony with previous results of simple Gō-like and experimental analysis, supporting that the folding route of SH3 domain is robust and coded by the native topology. The hybrid method provides useful tools for analyzing roles of physical interactions in determining folding mechanisms.     

A kinetic study on the reductive opening of the diphenylmethylene acetal in methyl 2,3-O-diphenylmethylene-α-l-rhamnopyranoside

Reductive opening of the diphenylmethyl acetal in methyl 2,3-O-diphenylmethylene-α-l-rhamnopyranoside has been investigated by kinetic studies, and the results have been compared to those recently obtained by quantum chemical calculations. In contrast to the previous theoretical calculations which related only to the presumably rate limiting step of the reductive opening, the reaction system LiAlH₄, AlCl₃, and the title compound consists of at least four simultaneous reactions. Nevertheless, reasonable agreement can be found between the activation Gibbs free energy obtained from kinetic measurements and the theoretically calculated ones in spite of the experimental errors and the approximate nature of theoretical calculations.     

Probability density functions for axial ratios of sectioning profiles of anisotropically arranged elliptical microvessels

The article theoretically regards probability density functions (PDFs) for axial ratio (X/Y) of sectioning profiles of elliptical microvessels (MVs) arranged with anisotropy in a biological tissue volume. A technique for the PDF_X/Y calculations in anisotropy of the elliptical MVs is described. The essence of this technique is introducing anisotropy in PDF(α,φ), i.e. the function of the joint distribution of the polar and planar angles α and φ, which define mutual orientation of the elliptical MVs and sectioning planes. With the aid of this technique, the anisotropy cases are studied with PDF(α,φ) given by pair combinations of the following distributions: (i) a uniform distribution of the angles α and/or φ, (ii) the angle α distribution with , and (iii) Gaussian distributions of the α or φ values. Specifically, PDF_X/Y curves are obtained for MVs with the true, or three-dimensional, axial ratio X₀/Y₀=2.0, and the anisotropy effects on the X/Y expected frequencies are analysed. Conclusions of this analysis, the PDF_X/Y calculation technique, and the PDF_X/Y curves obtained are useful for stereological reconstruction of anisotropically organised microcirculatory networks, with an ellipticity of their MVs being taken into consideration.     

Synthesis of 3-O Substituted D-Mannoses

1,2,4,6-Tetra-O-acetyl-3-O-benzyl-α-D-mannopyranose (7) was obtained in good yield from 3,4,6-tri-O-benzyl-1,2-O-(1-methoxyethylidene)-β-D-mannopyranose (1) by acetolysis. Hydrogenolysis of 7 afforded 1,2,4,6-tetra-O-acetyl-α-D-mannopyranose which is a versatile intermediate for the preparation of other 3-O-substituted D-mannoses, such as 3-O-methyl-D-mannose and 3-O-α-D-mannopyranosyl-D-mannose. 3,4-Di-O-methyl-D-mannose was readily prepared from 1,2,6-tri-O-acetyl-3,4-di-O-benzyl-α-D-mannopyranose, which was also obtained from 1 by controlled acetolysis.     

Applying Canonical Analysis to a Kind of Generalized Regression

This paper is concerned with the generalized model E(φ(Y)! X) =φ(X) involving the transformations on both the predictor vector X and the response variable Y. For this purpose, Taylor expansions and canonical analysis are applied. For optimizing the expansions, it is shown by a simulation study that not only prediction error, the combination of model error and noise error, is an important index, but the distribution of the residuals and the t-values of the coefficients also must be considered. Furthermore, the results of penicillin titrition show that the practical situations often need to be considered in selecting an appropriate model for a real-life problem.     

Folding of spectrin's SH3 domain in the presence of spectrin repeats

The multifunctional protein spectrin contains several different structural motifs, such as spectrin repeats and a SH3 domain. Both triple-helix spectrin repeats and the SH3 domain are believed to form independent structural entities. In alpha-spectrins the SH3 domain is localized to repeat 9, where it is positioned between helix B and helix C in the repeat unit. The presence of SH3 in repeat 9 decreases the thermal stability considerably of this repeat unit while another insert in helix C does not seem to affect the stability. Addition of one or two adjacent repeat units increases the thermal stability from ca 25 degrees C to 41 and 48 degrees C, respectively. Despite the differences in thermal stability, the folding properties of peptides comprising the SH3 domain only or together with one or more repeats are more or less the same.     

Enzyme reactions in a membrane model. 1. A new technique to study enzyme reactions in monolayers

Enzymic hydrolysis by pancreatic phospholipase A (EC 3.1.1.4) of 3-sn-didodecanoyl phosphatidylglycerol was studied under constant surface pressure. A technical device is described which automatically keeps the surface pressure constant and which continuously registers the movement of the surface barrier. A new trough design is proposed which consists of two compartments connected by a narrow surface canal. With this trough the recorded kinetic plots are linear, in contrast to the non-linear kinetic plots obtained with the usual one-compartment trough.     

Synthese der “repeating unit” der O-spezifischen kette des lipopolysaccharides aus Aeromonas salmonicida

8-Methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-β-d-mannopyranoside reacted with 2,3,4-tri-O-acetyl-α-l-rhamnopyranosyl bromide to give a disaccharide from the which the glycosyl-acceptor 8-methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2,4,-di-O-acetyl-α-l-rhamnopyranosyl)-β-d-manno pyranoside (19) was obtained. This glycosyl-acceptor with 2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl chloride to give trisaccharide derivative 22 and with 2,3,6-tri-O-(α-²H₂)benzyl-4-O-(2,3,4,6-tetra-O-(α-²H₂)benzyl-α-d-glucopyranosyl)-α-d-glucopyranosyl chloride to give tetrasaccharide derivative 29. Deblocking of 22 yielded 8-methoxycarbonyloctyl O-(α-d-glucopyranosyl)-(1→3)-O-α-l-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside and deblocking of 29 8-methoxycarbonyloctyle O-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl- (1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside. Both oligosaccharides represent the “repeating unit” of the O-specific chain of the lipopolysaccharide from Aeromonas salmonicida.