Proteins

The diiron-containing YtfE protein in Escherichia coli is pivotal in counteracting nitrosative stress, a critical barrier to bacterial viability. This study delves into the biochemical YtfE's conversion of nitric oxide to nitrous oxide, a for alleviating nitrosative stress. Through site-directed mutagenesis, we explored YtfE's molecular structure, with a particular focus on two internal transport tunnels important for its activity. Our findings illuminate Tunnel 1 as the primary conduit for substrate transport, regulated by conformational shifts within the N-terminal domain that enable substrate access to the diiron core in the C-terminal domain. Furthermore, Tunnel 2 emerges as a secondary, supportive route, activated when Tunnel 1 is compromised. This result challenges the previous model of distinct tunnels for substrate entry and product exit, suggesting both tunnels are capable of transporting substrates and products. Our engineering efforts enhanced the role of Tunnel 2, enabling a synergistic operation with Tunnel 1 and tripling YtfE's enzymatic activity compared to its wild-type form. This research not only deepens our understanding of YtfE’s regulatory mechanism for NO reduction but also introduces a strategy to amplify its enzymatic efficiency. The outcomes of our study have far-reaching implications, opening new paths for modulating bacterial stress responses and contributing to the ongoing development of bacterial strain engineering in denitrification processes.

Pulsed dipolar spectroscopy, such as double electron-electron resonance (DEER), has been underutilized in protein structure determination, despite its ability to provide valuable spatial information. In this study, we present DEERefiner, a user-friendly MATLAB-based GUI program that enables the modeling of protein structures by combining an initial structure and DEER distance restraints. We illustrate effectiveness of DEERefiner by successfully modeling the ligand-dependent conformational changes of the proton-drug antiporter LmrP to an extracellular-open-like conformation with an impressive precision of 0.76 Å. Additionally, DEERefiner was able to uncover a previously hypothesized but experimentally unresolved proton-dependent conformation of LmrP, characterized as an extracellular-closed/partially intracellular-open conformation, with a precision of 1.16 Å. Our work not only highlights the ability of DEER spectroscopy to model protein structures but also reveals the potential of DEERefiner to advance the field by providing an accessible and applicable tool for precise protein structure modeling, thereby paving the way for deeper insights into protein function.

Double electron-electron resonance (DEER) is a powerful technique for studying protein conformations. To preserve the room temperature ensemble, proteins are usually shock frozen in liquid nitrogen prior to DEER measurements. The use of cryoprotectant additives is, therefore, necessary to ensure the formation of vitrified state. Here, we present a simple modification of the freezing process by utilizing a flexible fused silica microcapillary, which increases the freezing rates and thus enables DEER measurement without the use of cryoprotectants. The Bid protein, which is highly sensitive to cryoprotectant additives, is used as a model. We show that DEER with the simple modification can successfully reveal the cold denaturation of Bid, which was not possible with the conventional DEER preparations. The DEER result reveals the nature of Bid folding. Our method advances DEER for capturing the chemically and thermally induced conformational changes of a protein in a cryoprotectant-free medium.

Understanding how proteins retain structural stability is not only of fundamental importance in biophysics but also critical to industrial production of antibodies and vaccines. Protein stability is known to depend mainly on two effects: internal hydrophobicity and H-bonding between protein surface and solvent. A challenging task is to identify their individual contributions to a protein. Here we investigate the structural stability of the apoptotic Bid protein in solutions containing various concentrations of guanidinium hydrochloride and urea using a combination of recently developed methods including the QTY (glutamine, threonine, and tyrosine) code and electron spin resonance (ESR)-based peak-height analysis. We show that when the internal hydrophobicity of Bid is broken down using the QTY code, the surface H-bonding alone is sufficient to retain the structural stability intact. When the surface H-bonding is disrupted, Bid becomes sensitive to the temperature-dependent internal hydrophobicity such that it exhibits a reversible cold unfolding above water’s freezing point. Using the combined approach, we show that the free-energy contributions of the two effects can be more reliably obtained. The surface H-bonds is more important than the other effect in determining the structural stability of Bid protein.