Synonymous Mutations Can Alter Protein Dimerization Through Localized Interface Misfolding Involving Self-entanglements.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

How synonymous mutations alter enzyme structure and function over long timescales.

The specific activity of enzymes can be altered over long timescales in cells by synonymous mutations that alter a messenger RNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we use multiscale modelling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B and dihydrofolate reductase) to understand experimentally measured changes in specific activity due to synonymous mutations. The modelling involves coarse-grained simulations of protein synthesis and post-translational behaviour, all-atom simulations to test robustness and quantum mechanics/molecular mechanics calculations to characterize enzymatic function. We show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. These long-lived states exhibit reduced catalytic activity, as shown by their increased activation energies for the reactions they catalyse.

A proteome-wide map of chaperone-assisted protein refolding in a cytosol-like milieu.

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain's intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis-mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.

Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less functional.

Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.

Near-Infrared and Visible Photoactivation to Uncage Carbon Monoxide from an Aqueous-Soluble PhotoCORM.

Multiphoton excitation allows one to access high energy excited states and perform valuable tasks in biological systems using tissue penetrating near-infrared (NIR) light. Here, we describe new photoactive manganese tricarbonyl complexes incorporating the ligand 4'-p-N,N-bis(2-hydroxyethyl)amino-benzyl-2,2':6',2″-terpyridine (TPYOH), which can serve as an antenna for two photon NIR excitation. Solutions of Mn(CO)3(TPYOH)X (X = Br- or CF3SO3-) complexes are very photoactive toward CO release under visible light excitation (405 nm, 451 nm). The same responses were also triggered by multiphoton excitation at 750 and 800 nm. In this context, we discuss the potential applications of these complexes as visible/NIR light photoactivated carbon monoxide releasing moieties (photoCORMs). We also report the isolation and crystal structures of the TPYOH complexes Mn(TPYOH)Cl2 and [Mn(TPYOH)2](CF3SO3)2, to illustrate a possible photolysis product(s).