Reaction path analysis from potential energy contributions using forces: An accessible estimator of reaction coordinate adequacy

The calculation of potential energy and free-energy profiles along complex chemical reactions or rare event processes is of great interest because of their importance for many areas in chemistry, molecular biology, and material science. One typical way to generate these profiles is to add a bias potential to modify the energy surface, which can act on a selected degree of freedom in the system. However, in these cases, the quality of the result is strongly dependent on the selection of the degree of freedom over which this bias potential acts. The present work introduces a simple method for the analysis of the degree of freedom selected to describe a chemical process. The proposed methodology is based on the decomposition of contributions to the potential energy profiles by the integration of forces along a reaction path, which allows evaluating the different contributions to the energy change. This could be useful for discriminating the contributions to the energy arising from different regions of the system, which is particularly useful in systems with complex environments that must be represented using hybrid quantum mechanics/molecular mechanics schemes. Furthermore, this methodology allows in generating a quick and simple analysis of the degree of freedom which is used to describe the potential energy profile associated with the reactive process. This is computationally more accessible than the corresponding free-energy profile and can therefore be used as a simple estimator of reaction coordinate adequacy.Fil: Foglia, Nicolás Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: González Lebrero, Mariano Camilo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Biekofsky, Rodolfo R.. Moebius Research Ltd.; Reino UnidoFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentin

Mapping Site-Specific Changes that Affect Stability of the NTerminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the 1H-15N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability

Cooperative cyclic interactions involved in metal binding to pairs of sites in EF-hand proteins

AbstractThis study focuses on a closed net of electron-pair donor–acceptor interactions, present in the core of all metal-bound EF-hand pairs, that link both metal ions across a short two-stranded β-sheet. A molecular model based on the above cycle of interactions was studied using semi-empirical molecular orbital quantum mechanical methods. The calculations indicate that the interactions in the model cycle are cooperative, that is, that the interaction energy of the cyclic structure is greater than that of the sum of isolated interactions between its components. The cooperativity in this cycle can be attributed to an increase in the stability of the interactions resulting from a mutual polarisation of the associated groups. The predicted polarisation of the amide groups in the cycle is in agreement with experimental NMR 15N deshielding observed for these amide groups upon metal binding. Experimental observations of strengthening of the β-sheet hydrogen bonds are also consistent with the model calculations. By this mechanism, the binding of the first metal ion would enhance the binding of the second metal ion, and thus, the intradomain cooperativity in cation binding of calmodulin and related EF-hand proteins can be ascribed, at least partly, to this short-range molecular mechanism

Unwinding the helical linker of calcium-loaded calmodulin: A molecular dynamics study

The fold of calmodulin (CaM) consists of two globular domains connected by a helical segment (the linker), whose conformational properties play a crucial role for the protein's molecular recognition processes. Here we investigate the structural properties of the linker by performing a 11.5 ns molecular dynamics (MD) simulation of calcium-loaded human CaM in aqueous solution. The calculations are based on the AMBER force field. The calculated S2 order parameters are in good accord with NMR data: The structure of the linker in our simulations is much more flexible than that emerging from the Homo sapiens X-ray structure, consistently with the helix unwinding observed experimentally in solution. This process occurs spontaneously in a nanosecond timescale, as observed also in a very recent simulation based on the GROMOS force field. A detailed description of the mechanism that determines the linker unwinding is provided, in which electrostatic contacts between the two globular domains play a critical role. The orientation of the domains emerging from our MD calculations is consistent both with former X-ray scattering data and a recent NMR work. Based on our findings, a rationale for the experimentally measured entropy cost associated to binding to the protein's cellular partners is also given

Conformational studies of the beta-subunit of the high affinity IgE receptor : circular dichroism and molecular modelling

The receptor with high affinity for immunoglobulin E (Fc epsilon RI) on mast cells and basophils plays an important role in mediating many of the pathophysiological phenomena associated with allergy. Fc epsilon RI is a tetrameric complex, alpha beta gamma2, of non-covalently attached subunits: one IgE-binding alpha-subunit with the binding site in the extracellular part of the chain, one beta-subunit and a dimer of disulphide linked gamma-subunits. In the present work, prediction of the three-dimensional structure of the four membrane-spanning segments of the beta-subunit has been achieved using rules of helix-helix packing arrangements and molecular dynamics calculations. It yielded a four-helix bundle with specific Van der Waals interactions between the helices. This four-helix bundle was used as a framework upon which to calculate the conformation of the beta-subunit excluding the C and N terminal cytoplasmic tails, but including the three chains that connect the four helices in the bundle. Separately, these synthetic 11, 17 and 29 residue bridge peptides were examined by circular dichroism (CD) spectroscopy and a degree of alpha-helical content in these bridge peptides was found. Additional molecular modelling of the bridge peptides indicate the central residues of these as the location of the helical moieties. Finally, in the model proposed for the beta-subunit, for each pair of consecutive transmembrane (TM) helices and its bridge peptide, a helix-loop-helix-loop-helix motif was found.Peer reviewe

Spectroscopy and modelling of the cytoplasmic domain of the gamma-subunit of the high affinity immunoglobulin E receptor

The high affinity receptor for IgE, Fc epsilon RI, is responsible for immediate hypersensitivity reactions. In rodents Fc epsilon RI is a tetrameric complex, alpha beta gamma 2 of non-covalently attached subunits: one IgE-binding alpha subunit with the binding site in the extracellular part of the chain, one beta-subunit and a dimer of disulphide linked gamma-subunits. Although there is an increasing evidence that the gamma-subunit chains are important signalling proteins that appear to function through a common Tyr-Leu-Tyr-Leu amino acid motif present in their cytoplasmic tails, which link the ligand binding specificity of their associated chains to signal transduction pathways, many questions related to conformation and function of this subunit remain to be answered. In the present work, the 36-residue cytoplasmic domain of the gamma-subunit has been synthesized and conformational studies by the combined use of Fourier transform infrared (FTIR), circular dichroism (CD) and nuclear magnetic resonance (NMR) have been performed. Based on the constraints found by these methods, conformational models of the cytoplasmic tail of the gamma-subunit are proposed and discussed.Peer reviewe