Deviation from the Residue-Based Linear Free Energy Relationship Reveals a Non-Native Structure in Protein Folding

Multiprobe measurements, such as NMR and hydrogen exchange study, can provide the equilibrium constant K and kinetic rate constant k of the structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between residue-specific log K values and residue-specific log k values for the two-state topological isomerization of a 27-residue peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific equilibrium and kinetic constants in various exchange processes, including protein folding, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of log-log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Protein molecules quickly fold into their native structures and change their conformations smoothly. Theoretical studies and molecular simulations advocate that the physicochemical basis is the consistency principle and the minimal frustration principle: Non-native structures/interactions are absent or minimized along the folding pathway. The linearity of the residue-based free energy relationship demonstrates experimentally the absence of non-native structures in transition states. In this context, the hydrogen exchange study of apomyoglobin folding intermediates is particularly interesting. We found that the residues that deviated from the linear relationship corresponded to the non-native structure, which had been identified by other experiments. The rbLFER provides a unique and practical method to probe the dynamic aspects of the transition states of protein molecules.

HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain's allostery and unfolding upon B12 and TonB binding

To import large metabolites across the outer membrane of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force that powers transport (1). But, the role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding (2) while others propose force-independent pore formation (3) by TonB binding leading to breakage of a salt bridge termed the "Ionic Lock". Our hydrogen exchange/mass spectrometry measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12 bound and the B12&TonB bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.

Radiochemical Studies of Hydrogen Exchange in Organic Compounds

<p>A property of a new or unknown organic compound which must be determined once the empirical formula and molecular weight are known, is the number of active or replaceable hydrogen atoms which the compound contains. These include hydrogen atoms present in amine, hydroxyl, carboxyl and other groups, where the hydrogen is not bound to a carbon atom but to an oxygen, nitrogen or sulphur atom or is in a position where it can ionize. The most general method by which this may be done quantitatively, is the one originally due to Zerewitinoff Zerewitinoff - Berichte 40 2023 (1907) 41 2233 (1908) 42 4802 (1909) 43 3590 (1910) 47 1659 (1914) 47 2417 (1914) and since developed on a micro scale by Roth A. Soltys Mikrochemie 20 107 (1936), Flaschentrager A. Roth Mikrochemie 11 140 (1932), whose method incorporates work by Tschugaeff - Flaschentrager z. Physiol Chem. 146 219 (1923) and the other two authors, and Soltys L. Tschugaeff Berichte 35 3912 (1902), and incorporates many of the latest improvements. This involves the quantatatively evolution of methane from reaction of the Grignard reagent MeMgI on groups such as -SH, -OH, -NH2, -COOH etc., i.e. those groups containing active or replaceable hydrogen atoms. Analysis by this method requires extreme care in technique and exact attention to experimental details. High results are obtained if the solvent or any part of the apparatus contains moisture and the whole determination must be carried out in an atmosphere of nitrogen to avoid reaction of the Grignard reagent with any oxygen present. Low results are obtained if the test solution does not dissolve completely in the chosen solvent and it is essential to carry out a blank prior to each analysis. The proceedure is labourious and painstaking and gives an accuracy of not greater than 5% using 3-5 mgm of organic compound. It also has the disadvantage that the Grignard Reagent will also react with other groups, such as carbonyl, aldehyde, nitrile etc., which may be present. This method cannot be applied to highly water soluble compounds which do not dissolve in ethers or other organic solvents and as the molecular size or complexity of the sample increases, the accuracy of the gasometric reactions becomes less, due to side reactions and incomplete reaction.</p>

Radiochemical Studies of Hydrogen Exchange in Organic Compounds

<p>A property of a new or unknown organic compound which must be determined once the empirical formula and molecular weight are known, is the number of active or replaceable hydrogen atoms which the compound contains. These include hydrogen atoms present in amine, hydroxyl, carboxyl and other groups, where the hydrogen is not bound to a carbon atom but to an oxygen, nitrogen or sulphur atom or is in a position where it can ionize. The most general method by which this may be done quantitatively, is the one originally due to Zerewitinoff Zerewitinoff - Berichte 40 2023 (1907) 41 2233 (1908) 42 4802 (1909) 43 3590 (1910) 47 1659 (1914) 47 2417 (1914) and since developed on a micro scale by Roth A. Soltys Mikrochemie 20 107 (1936), Flaschentrager A. Roth Mikrochemie 11 140 (1932), whose method incorporates work by Tschugaeff - Flaschentrager z. Physiol Chem. 146 219 (1923) and the other two authors, and Soltys L. Tschugaeff Berichte 35 3912 (1902), and incorporates many of the latest improvements. This involves the quantatatively evolution of methane from reaction of the Grignard reagent MeMgI on groups such as -SH, -OH, -NH2, -COOH etc., i.e. those groups containing active or replaceable hydrogen atoms. Analysis by this method requires extreme care in technique and exact attention to experimental details. High results are obtained if the solvent or any part of the apparatus contains moisture and the whole determination must be carried out in an atmosphere of nitrogen to avoid reaction of the Grignard reagent with any oxygen present. Low results are obtained if the test solution does not dissolve completely in the chosen solvent and it is essential to carry out a blank prior to each analysis. The proceedure is labourious and painstaking and gives an accuracy of not greater than 5% using 3-5 mgm of organic compound. It also has the disadvantage that the Grignard Reagent will also react with other groups, such as carbonyl, aldehyde, nitrile etc., which may be present. This method cannot be applied to highly water soluble compounds which do not dissolve in ethers or other organic solvents and as the molecular size or complexity of the sample increases, the accuracy of the gasometric reactions becomes less, due to side reactions and incomplete reaction.</p>

The Silicon–Hydrogen Exchange Reaction: Catalytic Kinetic Resolution of 2-Substituted Cyclic Ketones

We have recently reported the strong and confined, chiral acid-catalyzed asymmetric “silicon−hydrogen exchange reaction”. One aspect of this transformation is that it enables access to enantiopure enol silanes in a tautomerizing σ-bond metathesis, via deprotosilylation of ketones with allyl silanes as the silicon source. However, until today, this reaction has not been applied to racemic, 2-substituted, cyclic ketones. We show here that these important substrates readily undergo a highly enantioselective kinetic resolution furnishing the corresponding kinetically preferred enol silanes. Mechanistic studies suggest the fascinating possibility of advancing the process to a dynamic kinetic resolution.

Theoretical Investigation into the Solvent Effect on the Thermal Decomposition of RDX in Tetrahydrofuran, Acetone, Toluene and Benzene

In order to clarify the solvent effect on the thermal decomposition of explosive, the N–NO2 trigger-bond strengths and ring strains of RDX (cyclotrimethylenetrinitramine) in its H-bonded complexes with solvent molecules (i.e., tetrahydrofuran, acetone, toluene and benzene), and the activation energies of the intermolecular hydrogen exchanges between the solvent molecules and C3H8O2N4 or CH4O2N2, as the model molecule of RDX, were investigated by the BHandHLYP, B3LYP, MP2(full) and M06-2X methods with the 6-311++G(2df,2p) basis set, accompanied by a comparison with the calculations by the integral equation formalism polarized continuum model. The solvent effects ignore the ring strain while strengthen the N–NO2 bond, leading to a decreased sensitivity, as is opposite to the experimental results. However, the activation energies are in the order of C3H8O2N4/CH4O2N2∙∙∙acetone < C3H8O2N4/CH4O2N2∙∙∙THF < C3H8O2N4/CH4O2N2∙∙∙toluene < C3H8O2N4/CH4O2N2∙∙∙benzene < C3H8O2N4/CH4O2N2, suggesting that the order of the critical explosion temperatures should be RDX∙∙∙acetone < RDX∙∙∙THF < RDX∙∙∙toluene < RDX∙∙∙benzene < RDX, as is roughly consistent with the experimental results. Therefore, the intermolecular hydrogen exchange with the HONO elimination is the essence of the solvent effect on the thermal decomposition of RDX. The solvent effect is confirmed by reduced density gradient, atoms in molecules and surface electrostatic potentials.