Unified Description of Urea Denaturation: Backbone and Side Chains Contribute Equally in the Transfer Model

After studying protein denaturation by urea for many decades, conflicting views of the role of the side chains and the backbone have emerged; many results suggest that urea denatures by enhancing the solubility of both the side chains and the backbone, but the frequently applied transfer model (TM) so far ascribes denaturation exclusively to urea’s action on the backbone. We use molecular dynamics simulations to rigorously test one of the TM’s key assumptions, the proportionality of a molecule’s transfer free energy (TFE) and its solvent-accessible surface. The performance of the TM as it is usually implemented turns out to be unsatisfactory, but the proportionality is satisfied very well after an inconsistency in the treatment of the backbone contribution is corrected. This inconsistency has so far gone unnoticed as it was obscured by a compensating error in the side-chain group TFEs used so far. The revised “universal backbone” TM presented in this work shows excellent accuracy in the prediction of experimental <i>m</i> values of a set of 36 proteins. It also settles the conflicting views regarding the role of the side chains because it predicts that both the side chains and the backbone on average contribute favorably to denaturation by urea

Nuclear Inelastic Scattering and Mössbauer Spectroscopy as Local Probes for Ligand Binding Modes and Electronic Properties in Proteins: Vibrational Behavior of a Ferriheme Center inside a β-Barrel Protein

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN^–), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect <i>Rhodnius prolixus</i>. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2–NO complex and Type I and II electronic ground states of the NP2–CN^– and NP2–histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN^– and histamine, might be a consequence of the protonation state of the heme carboxyls

Nuclear Inelastic Scattering and Mössbauer Spectroscopy as Local Probes for Ligand Binding Modes and Electronic Properties in Proteins: Vibrational Behavior of a Ferriheme Center inside a β-Barrel Protein

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN^–), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect <i>Rhodnius prolixus</i>. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2–NO complex and Type I and II electronic ground states of the NP2–CN^– and NP2–histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN^– and histamine, might be a consequence of the protonation state of the heme carboxyls