Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways and complex proteinaceous machinery at the plastid surface to direct these proteins back to their target organelle. Chloroplasts are the most well-characterized plastids, responsible for photosynthesis and other important metabolic functions. The biogenesis and function of chloroplasts rely heavily on the fidelity of intracellular protein trafficking pathways. Therefore, understanding these pathways and their regulation is essential. Furthermore, the chloroplast outer membrane proteome remains relatively uncharted territory in our understanding of protein targeting. Many key players in the cytosol, receptors at the organelle surface, and insertases that facilitate insertion into the chloroplast outer membrane remain elusive for this group of proteins. In this review, we summarize recent advances in the understanding of well-characterized chloroplast outer membrane protein targeting pathways as well as provide new insights into novel targeting signals and pathways more recently identified using a bioinformatic approach. As a result of our analyses, we expand the known number of chloroplast outer membrane proteins from 117 to 138.

In microbial biofilms, bacterial cells are encased in a self-produced matrix of polymers (e.g., exopolysaccharides) that enable surface adherence and protect against environmental stressors. For example, the wrinkly spreader phenotype of Pseudomonas fluorescens colonizes food/water sources and human tissue to form robust biofilms that can spread across surfaces. This biofilm largely consists of bacterial cellulose produced by the cellulose synthase proteins encoded by the wss (WS structural) operon, which also occurs in other species, including pathogenic Achromobacter species. Although phenotypic mutant analysis of the wssFGHI genes has previously shown that they are responsible for acetylation of bacterial cellulose, their specific roles remain unknown and distinct from the recently identified cellulose phosphoethanolamine modification found in other species. Here, we have purified the C-terminal soluble form of WssI from P. fluorescens and Achromobacter insuavis and demonstrated acetylesterase activity with chromogenic substrates. The kinetic parameters (k_cat/K_M values of 13 and 8.0 M^-1 s^-1, respectively) indicate that these enzymes are up to four times more catalytically efficient than the closest characterized homolog, AlgJ from the alginate synthase. Unlike AlgJ and its cognate alginate polymer, WssI also demonstrated acetyltransferase activity onto cellulose oligomers (e.g., cellotetraose to cellohexaose) with multiple acetyl donor substrates (p-nitrophenyl acetate, 4-methylumbelliferyl acetate, and acetyl-CoA). Finally, a high-throughput screen identified three low micromolar WssI inhibitors that may be useful for chemically interrogating cellulose acetylation and biofilm formation.

Chloroplasts, the essential organelles responsible for photosynthesis, rely on the coordinated import of nuclear-encoded proteins for their biogenesis and function. TOC159 receptors are critical components of the translocon at the outer membrane of chloroplasts (TOC complex), responsible for recognizing and importing preproteins. However, the mechanisms by which TOC159 itself is targeted and integrated into the chloroplast outer envelope are only partially understood. In this study, we explore the structural aspects of the C-terminal membrane (M) domain of Arabidopsis thaliana TOC159, with a focus on its role in chloroplast outer envelope targeting and TOC complex integration. Structural predictions using AlphaFold and far-UV circular dichroism (CD) spectroscopy experiments suggested that the M domain forms an integral β-barrel that fuses with the β-barrel of TOC75, forming a hybrid channel at the chloroplast outer envelope. We identified a novel bi-partite targeting signal within the M domain, composed of a C-terminal β-signal with the consensus sequence G-Q-Φ-[ST]-Φ-[RK]-X-[SN]-[ST] and a transit peptide (TP)-like sequence. Using fluorescence microscopy and mutational analyses, we demonstrated that the β-signal is essential for chloroplast targeting, while the TP-like sequence enhances targeting ejiciency. Furthermore, CD spectroscopy and Langmuir-Blodgett trough experiments revealed that the TP-like sequence preferentially interacts with galactolipids unique to the chloroplast outer membrane, particularly sulfoquinovosyl diacylglycerol (SQDG), facilitating chloroplast outer envelope targeting specificity. Overall, our findings provide a deeper understanding of TOC159 receptor targeting mechanisms, TOC complex biogenesis and highlight the broader significance of lipid-mediated targeting in intracellular protein trajicking. These results pave the way for future studies on the role of TP-like sequences in chloroplast outer membrane protein targeting and other novel targeting pathways more generally.

Uncoupling proteins (UCPs) are members of the mitochondrial carrier family (MCF) that transport protons across the inner mitochondrial membrane, thereby uncoupling electron transport from ATP synthesis. The stoichiometry of UCPs, and the possibility of co-existence of this protein as mono-meric and associated forms in lipid membranes remain an intriguing open question. In the current study, the tertiary structure of UCP2 was analyzed both experimentally and through molecular dynamics (MD) simulations. After recombinant expression of UCP2 in the inner membrane of E. coli , the protein was directly extracted from the bacterial membranes with a non-denaturing detergent and purified both as a pure monomer and as a mixture of monomers, dimers and tetramers. Both protein preparations were re-constituted in egg yolk lipid vesicles. Gel electrophoresis, circular dichroism spectroscopy and fluorescence methods were used to characterize the structure and the proton transport function of protein. UCP2 showed unique stable tetrameric forms in lipid bilayers. MD simulations using membrane lipids and principal component analysis support the experimental results and provided new molecular insights into the nature of noncovalent interactions in oligomeric UCP2. MD simulations indicate that UCP2 tetramers are asymmetric dimers of dimers, in which the interactions between the monomers forming the dimer are stronger than the interactions between the dimers within the tetramer. It is also shown that UCP2 has a specific tendency to form functional tetramers in lipid bilayers, capable of proton transport. The asymmetric nature of the UCP2 tetramer could act as a scaffold for regulating the activity of the monomeric units through cooperative intercommunication between these subunits. Under similar experimental conditions, the structurally comparable ADP/ATP carrier protein did not form tetramers in vesicles, implying that spontaneous tetramerization cannot be generalized to all MCF members. STATEMENT OF SIGNIFICANCE: Self-assembly of membrane proteins plays a significant role in their biological function. In this article, both experimental and computational evidence are provided for spontaneous tetramerization of one of the mitochondrial uncoupling proteins (UCP2) in model lipid membranes. It is also shown that the tetrameric form of UCP2 is capable of proton transport, which leads to regulation of ATP synthesis in mitochondrion. Molecular dynamics simulations confirm the presence of asymmetric UCP2 tetramers as a potential scaffold for regulating the activity of the monomeric units through mutual intercommunication. The outcome of this study provides a solid ground for potential co-existence of monomeric and multimeric functional forms of UCPs that contributes to a deeper molecular insight into their structure and function.

The generation of high-performance enzyme variants with desired physicochemical and functional properties presents a formidable challenge in the field of protein engineering. Existing in silico design methods are limited by inadequate training data, insufficient diversity within datasets, and suboptimal sampling techniques. Here, we introduce a novel approach that addresses these limitations and significantly improves the efficiency of generating functional enzyme variants. Using a multimodal approach, NeuroFold can leverage sequence, structural, and homology data during both sampling and discrimination phases, thereby enabling more diverse and informed sampling of the sequence space. Our model demonstrated a 40-fold increase in Spearman rank correlation as compared to large language models (LLMs) such as ESM-1v and empowers the rapid creation of high-quality enzyme variants, such as the β-lactamase variants generated by NeuroFold in this study, which demonstrated increased thermostability and varying levels of activity. This pipeline represents a promising advancement in the field of enzyme engineering, offering a valuable tool for the development of novel enzymes with enhanced performance and desired chemical properties.