Size fractionation of thermal aggregates of immunoglobulin G

Purified pooled human immunoglobulin G (IgG) in solution, when extensively heated at high temperatures or for long periods, irreversibly aggregates and insoluble precipitates result. However, when IgG solutions are heated in the temperature range 55-65 degrees C for more limited time periods, soluble turbid polydispersed aggregate mixtures are obtained. Gel filtration of such aggregate mixtures on calibrated Bio-Rad A-150m columns demonstrates a continuous size distribution from dimers to aggregates as large as 4 X 10(7) Da (200-mers) with no particular size predominant. Chromatographically reproducible cuts of narrow size heterogeneity can be obtained by short-time fraction collection. Elution-time reproducibility is excellent both for mixture and for individual cuts. Stability studies indicate that reproducible and stable aggregates may be made from purified IgG and that fractionated aggregates should be stored quick-frozen until needed. Sized IgG aggregates have proved useful in reactivity studies with rheumatoid factor, animal anti-IgG antibodies, and complement.     

基于蛋白质二级结构热稳定性的正交方法

对蛋白质热稳定性的研究是解析蛋白高级结构,开发蛋白功能及新药物研发过程中的一个重要环节,是对其结构分析的一个重要关切点.观测蛋白质的圆二色光谱随温度程序变化而改变是研究其热稳定性的常用手段,传统的实验方法为选用某一单波长作为测试点,通过连续升温测试蛋白在单波长下的圆二色变温曲线,然后拟合出Tm值,此方法所得的信息有限,...     

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state.     

Crystal structure and structural stability of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3

To elucidate the structural basis for the high stability of acylphosphatase (AcP) from Pyrococcus horikoshii OT3, we determined its crystal structure at 1.72 A resolution. P. horikoshii AcP possesses high stability despite its approximately 30% sequence identity with eukaryotic enzymes that have moderate thermostability. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic counterparts. The crystal structure of P. horikoshii AcP shows the same fold betaalphabetabetaalphabeta topology and the conserved putative catalytic residues as observed in eukaryotic enzymes. Comparison with the crystal structure of bovine common-type AcP and that of D. melanogaster AcP (AcPDro2) as representative of eukaryotic AcP revealed some significant characteristics in P. horikoshii AcP that likely play important roles in structural stability: (1) shortening of the flexible N-terminal region and long loop; (2) an increased number of ion pairs on the protein surface; (3) stabilization of the loop structure by hydrogen bonds. In P. horikoshii AcP, two ion pair networks were observed one located in the loop structure positioned near the C-terminus, and other on the beta-sheet. The importance of ion pairs for structural stability was confirmed by site-directed mutation and denaturation induced by guanidium chloride.     

Inhibition of murine N1-acetylated polyamine oxidase by an acetylenic amine and the allenic amine, MDL 72527

Murine N¹-acetylated polyamine oxidase (mPAO) was treated with N,N′-bis-(prop-2-ynyl)-1,4-diaminobutane, a poor substrate and inhibitor for the enzyme, with K_m and K_i values in the millimolar range. Apparently, its oxidation produces prop-2-ynal, which reacts with amino acyl nucleophiles. Using a steady-state kinetic assay, four phases were identified, the first being the oxidation of the compound via Michealis-Menten-type kinetics. As prop-2-ynal accumulates, there is a biphasic reduction in the rate. This process leads to an mPAO form that is nearly inactive (fourth phase), but displays classical Michealis-Menten-type kinetics. The enzyme-bound flavin is not modified in this process. In contrast, micromolar concentrations of the MDL 72527 (N,N′-bis-[buta-2,3-dienyl]-1,4-diaminobutane) inhibited mPAO rapidly and completely. It inhibits by first binding tightly and apparently irreversibly, and then slowly converts to a species where the inhibitor is covalently bound to the N5-position of the flavin’s isoalloxazine ring. The covalent adduct was identified as a flavocyanine.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

Statistical mechanics of protein folding by cluster distance geometry

This is our second type of model for protein folding where the configurational parameters and the effective potential energy function are chosen in such a way that all conformations are described and the canonical partition function can be evaluated analytically. Structure is described in terms of distances between pairs of sequentially contiguous blocks of eight residues, and all possible conformations are grouped into 71 subsets in terms of bounds on these distances. The energy is taken to be a sum of pairwise interactions between such blocks. The 210 energy parameters were adjusted so that the native folds of 32 small proteins are favored in free energy over the denatured state. We then found 146 proteins having negligible sequence similarity to any of the training proteins, yet the free energy of the respective correct native states were favored over the denatured state.     

The regulatory factor SipA is a highly stable β-II class protein with a SH3 fold

The small regulator SipA, interacts with the ATP-binding domain of non-bleaching sensor histidine kinase (NblS), the most conserved histidine kinase in cyanobacteria. NblS regulates photosynthesis and acclimation to a variety of environmental conditions. We show here that SipA is a highly stable protein in a wide pH range, with a thermal denaturation midpoint of 345 K. Circular dichroism and 1D ¹H NMR spectroscopies, as well as modelling, suggest that SipA is a β-II class protein, with short strands followed by turns and long random-coil polypeptide patches, matching the SH3 fold. The experimentally determined m-value and the heat capacity change upon thermal unfolding (ΔC_p) closely agreed with the corresponding theoretical values predicted from the structural model, further supporting its accuracy.     

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.