Exploring the complex molecular processes of mitochondrial protein import, this study focuses on the TOM complex and chaperones, employing advanced techniques such as Cryo-EM. The research provides an attempt to high-resolution 3D reconstruction of the yeast chaperone complex Hsp70-Djp1, unveiling crucial insights into mitochondrial precursor protein processing. Additionally, investigations into Human-Tom34 interactions with various solubilization tags reveal distinct specificity patterns. However, the study highlights temporal dynamics in the complex formation of Human-Tom34, Hsp90, and Hsc70StrepII, emphasizing the necessity of a multifaceted approach to comprehend protein-protein interactions. These findings contribute valuable knowledge to mitochondrial biology, potentially informing therapeutic targets for related disorders.

Nitric oxide (NO) signaling in mammals occurs through the activation of soluble guanylate cyclase (sGC), which results in the stimulation of cGMP mediated signaling pathways important for blood vessel homeostasis and neurotransmission. sGC is a heterodimeric hemoprotein comprised, typically, of alpha1 and beta1 subunits, with the heme cofactor responsible for selective NO binding located at the N-terminus of the beta1 subunit. The heme domain of sGC belongs to a larger class of proteins termed Heme-Nitric oxide/OXygen binding (H-NOX) domains, named as such to encompass their ability to serve as either NO or oxygen sensors. Functional characterization of H-NOX proteins from prokaryotes has provided important insight into the roles these proteins serve in biological contexts. However, fundamental questions remain about the mechanisms of NO activation and signal transduction within H-NOX signaling pathways.

Extensive studies on several members of the H-NOX family have established the functional importance of topological features within the protein scaffold that tune the electronic properties of the heme. However, there is little knowledge regarding the functional role of H-NOX protein structure in modulating gas diffusion as a means for tuning the ligand-binding properties. Using X-ray crystallography with xenon as a probe for gas diffusion pathways, a bifurcated tunnel network between the solvent and interior heme site was mapped in a prokaryotic H-NOX protein. Site-directed mutagenesis and kinetic measurements demonstrate that blocking the tunnels to hinder gas diffusion has important consequences on gas-binding affinity. These data suggest that this tunnel network in H-NOX proteins may serve functional roles in controlling the flux of ligands important for tuning gas-mediated signaling.

A key molecular event during NO-induced activation of H-NOX proteins is loss of the heme-histidine bond and formation of a five-coordinate nitrosyl complex. Although this has been known for quite some time, molecular details into this process have remained elusive. Using X-ray crystallography, structures of a prokaryotic H-NOX protein in the ferrous-unliganded as well as both six-coordinate and five-coordinate nitrosyl complexes were reported. These structures show that several features in the unliganded state maintain the heme in a distorted conformation that relaxes towards planarity following NO binding and loss of the heme-histidine bond. Because the heme and protein conformations are intimately coupled, relaxation of the heme towards planarity results in a pronounced conformational change in the H-NOX protein involving a rotational displacement of the distal subdomain about the proximal subdomain. It is hypothesized that this conformational change is the method by which H-NOX proteins communicate NO binding to downstream signaling partners.

The observation that H-NOX proteins can bind NO as five-coordinate complexes with NO located in either the distal or proximal heme pockets presented the possibility that these two nitrosyl complexes would yield different NO signaling lifetimes as a result of their intrinsically different dissociation rates. To test this hypothesis, two different chemical based NO traps were employed to measure the dissociation rate constants from distal and proximal-bound, five-coordinate NO complexes in a prokaryotic H-NOX protein. Unexpectedly, under all conditions tested, similar rate constants for NO dissociation are observed between these two nitrosyl species. As such, it is hypothesized that the rate-determining step for dissociation of NO from both five-coordinate NO species is loss of the NO-iron bond.

Prokaryotic H-NOX proteins are often found in predicted operons with putative signaling proteins, predominantly histidine kinases. Importantly, histidine kinases are commonly part of two-component signaling systems, which serve as a basic stimulus-response pathway critical for prokaryotes to sense and respond to extracellular stimuli. Furthermore, it has been established that the H-NOX: histidine kinase signaling couple from Shewanella oneidensis regulates the motility of the organism in response to NO. To obtain insight into the mechanism by which H-NOX proteins regulate histidine kinase autophosphorylation upon binding NO, various biophysical techniques were employed to determine the architecture of the H-NOX: histidine kinase complex from S. oneidensis. From these data, it is hypothesized that the NO-induced conformational change in the H-NOX protein allosterically inhibits histidine kinase activity by eliciting a series of conformational changes in the histidine kinase that places the phospho-accepting histidine in an inaccessible conformation.

Nitrogenase is a microbial enzyme critical for nitrogen fixation, catalyzing the reduction of dinitrogen (N₂) to ammonia (NH₃). This process is vital for the biosynthesis of nucleic acids and amino acids, contributing to half of global ammonia production. Despite extensive research, the mechanisms of substrate reduction, protein interaction, and ATP-dependent energy transduction remain unclear. Recently, cryogenic electron microscopy (cryoEM) has provided significant structural information on nitrogenase, although its oxygen-sensitive nature poses substantial challenges for this method.This study employs cryoEM to investigate the protein interactions within homologous and heterologous nitrogenase complexes. An optimized sample vitrification method was developed specifically for studying nitrogenase by cryoEM. Using this approach, we structurally characterized one homologous and two heterologous nitrogenase complexes. The electron microscopy (EM) density data, coupled with biochemical assays, revealed that while homologous and heterologous nitrogenase complexes share similar conformations in the activated complex state, they exhibit varied catalytic efficiencies and energy consumption rates. This study lays the groundwork for future cryoEM investigations of nitrogenase and provides important insights into its protein interactions, potentially guiding efforts to enhance the efficiency of biological nitrogen fixation.

There does not currently exist a standardized indicator of how well cryo-EM-derived models represent the density from which they were generated. We present a straightforward methodology that utilizes freely available tools to generate a suite of independent models and to evaluate their convergence in an EM density. These analyses provide both a quantitative and qualitative assessment of the precision of the models and their representation of the density, respectively, while concurrently providing a platform for assessing both global and local EM map quality. We further use standardized datasets to provide an expected deviation within a suite of models refined against EM maps reported to be at 5 Å resolution or better. Associating multiple atomic models with a deposited EM map provides a rapid and accessible reporter of convergence, a strong indicator of highly resolved molecular detail, and is an important step toward an FSC-independent assessment of map and model quality.