Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (¹⁰Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein–protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods. Figure

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Artificial neural system modeling of Monte Carlo simulations of polymers

In this work, a neural network was used to learn features in potential energy surfaces and relate those features to conformational properties of a series of polymers. Specifically, we modeled Monte Carlo simulations of 20 polymers in which we calculated the characteristic ratio and the temperature coefficient of the characteristic ratio for each polymer. We first created 20 rotational potential energy surfaces using MNDO procedures and then used these energy surfaces to produce 10000 chains, each chain 100 bonds long. From these results we calculated the mean-square end-to-end distance, the characteristic ratio and its corresponding temperature coefficient. A neural network was then used to model the results of these Monte Carlo calculations. We found that artificial neural network simulations were highly accurate in predicting the outcome of the Monte Carlo calculations for polymers for which it was not trained. The overall average error for prediction of the characteristic ratio was 4,82%, and the overall average error for prediction of the temperature coefficient was 0,89%.     

Binding,Release and Functionalization of Intact Pnictogen Tetrahedra Coordinated to Dicopper Complexes

The bridging MeCN ligand in the dicopper(I) complexes [(DPFN)Cu₂(μ,η¹ : η¹-MeCN)][X]₂ (X=weakly coordinating anion, NTf₂ ( 1 a ), FAl[OC₆F₁₀(C₆F₅)]₃ ( 1 b ), Al[OC(CF₃)₃]₄ ( 1 c )) was replaced by white phosphorus (P₄) or yellow arsenic (As₄) to yield [(DPFN)Cu₂(μ,η² : η²-E₄)][X]₂ (E=P ( 2 a – c ), As ( 3 a – c )). The molecular structures in the solid state reveal novel coordination modes for E₄ tetrahedra bonded to coinage metal ions. Experimental data and quantum chemical computations provide information concerning perturbations to the bonding in coordinated E₄ tetrahedra. Reactions with N-heterocyclic carbenes (NHCs) led to replacement of the E₄ tetrahedra with release of P₄ or As₄ and formation of [(DPFN)Cu₂(μ,η¹ : η¹-^MeNHC)][X]₂ ( 4 a,b ) or to an opening of one E−E bond leading to an unusual E₄ butterfly structural motif in [(DPFN)Cu₂(μ,η¹ : η¹-E₄^DippNHC)][X]₂ (E=P ( 5 a,b ), E=As ( 6 )). With a cyclic alkyl amino carbene (^EtCAAC), cleavage of two As−As bonds was observed to give two isomers of [(DPFN)Cu₂(μ,η² : η²-As₄^EtCAAC)][X]₂ ( 7 a,b ) with an unusual As₄-triangle+1 unit.     

Enhanced multi‐objective optimization of a dimpled channel through evolutionary algorithms and multiple surrogate methods

Using a multi‐objective evolutionary algorithm (MOEA) and enhanced surrogate approximations, the present study demonstrates the numerical analysis and optimization of staggered‐dimple channels. Two surrogates, the response surface approximation (RSA) model and the Kriging (KRG) model, are applied in light of the surrogate fidelity of the approximate analysis. An enhanced Pareto‐optimal front is obtained by performing local resampling of the Pareto‐optimal front, which provides relatively more accurate Pareto‐optimal solutions in the design space for each surrogate model. Three dimensionless design variables are selected, which are related to geometric parameters, namely, the channel height, dimple print diameter, dimple spacing, and dimple depth. Two objective functions are selected that are related to the heat transfer and pressure loss, respectively. The objective‐function values are numerically evaluated through Reynolds‐averaged Navier–Stokes analysis at the design points that are selected through the Latin hypercube sampling method. Using these numerical simulations two surrogates, viz, the RSA and Kriging models, are constructed for each objective function and a hybrid MOEA is applied to obtain the Pareto‐optimal front. For the particular implementation of surrogate models, it is observed that Pareto‐optimal predictions of the RSA model are better than those of the KRG model, whereas the KRG model predicts equally well at the off‐Pareto‐region (region away from the Pareto‐optimal solutions), which is not the case with the RSA model. The local resampling of the Pareto‐optimal front increases the fidelity of the approximate solutions near the Pareto‐optimal region. The ratios of the channel height to the dimple print diameter and of the dimple print diameter to the dimple pitch are found to be more sensitive along the Pareto‐optimal front than the ratio of the dimple depth to the print diameter. The decrease of the ratio of the channel height to the dimple diameter and the increase of the ratio of the dimple print diameter to the pitch lead to greater heat transfer at the expense of the pressure loss, whereas the ratio of the dimple depth to the print diameter is rather insensitive to Pareto‐optimal solutions. Pareto‐optimal solutions at higher values of the Nusselt number are associated with higher values of the pressure loss due to the increased recirculation, mixing of fluid and vorticity generation. Copyright © 2010 John Wiley & Sons, Ltd.     

Amphipols can support the activity of a membrane enzyme

Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations. However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent. In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced. PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent. All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.     

A compact rotating gravitational instanton

A compact rotating gravitational instanton (a positive-definite metric solution of the Einstein equations with Λ term) is presented. The manifold is the nontrivial S² fibre bundle over S² and has χ = 4, τ = 0, but no spinor structure. The metric can be obtained from a special limit of the positive-definite analytic extension of the Kerr-de Sitter metric or alternatively from the Taub-NUT metric with Λ term. The action is about $\text{4}\text{1}\text{2}\text{\%}$ less negative than that of the Einstein metric on the trivial bundle S² × S².     

Analysis of the click-evoked brainstem potentials in man unsing high-pass noise masking

Brainstem electrical responses (BSER) to 60-dB-SL click in noise high passed at various cutoff frequencies separated b 1/2-octave steps were recorded in normal-hearing adult subjects. By applying a derived response technique, narrow-band contributions to the BSER from specific portions of the basilar membrane were revealed. Latencies and amplitudes of the various waves in the derived BSER were recorded. Results indicate that nearly the whole cochlear partition can contribute to the brainstem response. The shifts in latency of waves I, III, and V and amplitude changes of waves I and III as a function of CF appear to be fully comparable to those of the AP. In contrast, the amplitude behavior of wave V as a function of CF is different from waves I and III depending upon frequency range. The discrepency in the behavior of wave V with respect to the earlier waves suggests some sort of neural reorganization at the level where was V is generated. The fact that there are contributions to the brainstem response from apical portions of the cochlea opens the possibility for extending the brainstem technique in assessing the higher cochlear turn function.