Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration.

Solution-state NMR typically requires 100 µM to 1 mM samples. This limitation prevents applications to mass-limited and aggregation-prone target molecules. Photochemically induced dynamic nuclear polarization was adapted to data collection on low-concentration samples by radiofrequency gating, enabling rapid 1D NMR spectral acquisition on aromatic amino acids and proteins bearing aromatic residues at nanomolar concentration, i.e., a full order of magnitude below other hyperpolarization techniques in liquids. Both backbone H1-C13 and side-chain resonances were enhanced, enabling secondary and tertiary structure analysis of proteins with remarkable spectral editing, via the 13C PREPRINT pulse sequence. Laser-enhanced 2D NMR spectra of 5 µM proteins at 600 MHz display 30-fold better S/N than conventional 2D data collected at 900 MHz. Sensitivity enhancements achieved with this technology, denoted as low-concentration photo-CIDNP (LC-photo-CIDNP), depend only weakly on laser intensity, highlighting the opportunity of safer and more cost-effective hypersensitive NMR applications employing low-power laser sources.

Critical Beginnings: Selective Tuning of Solubility and Structural Accuracy of Newly Synthesized Proteins by the Hsp70 Chaperone System.

Proteins are particularly prone to aggregation immediately after release from the ribosome, and it is therefore important to elucidate the role of chaperones during these key steps of protein life. The Hsp70 and trigger factor (TF) chaperone systems interact with nascent proteins during biogenesis and immediately post-translationally. It is unclear, however, whether these chaperones can prevent formation of soluble and insoluble aggregates. Here, we address this question by monitoring the solubility and structural accuracy of globin proteins biosynthesized in an Escherichia coli cell-free system containing different concentrations of the bacterial Hsp70 and TF chaperones. We find that Hsp70 concentrations required to grant solubility to newly synthesized proteins are extremely sensitive to client-protein sequence. Importantly, Hsp70 concentrations yielding soluble client proteins are insufficient to prevent formation of soluble aggregates. In fact, for some aggregation-prone protein variants, avoidance of soluble-aggregate formation demands Hsp70 concentrations that exceed cellular levels in E. coli. In all, our data highlight the prominent role of soluble aggregates upon nascent-protein release from the ribosome and show the limitations of the Hsp70 chaperone system in the case of highly aggregation-prone proteins. These results demonstrate the need to devise better strategies to prevent soluble-aggregate formation upon release from the ribosome.

Protein folding in vitro and in the cell: From a solitary journey to a team effort.

Correct protein folding is essential for the health and function of living organisms. Yet, it is not well understood how unfolded proteins reach their native state and avoid aggregation, especially within the cellular milieu. Some proteins, especially small, single-domain and apparent two-state folders, successfully attain their native state upon dilution from denaturant. Yet, many more proteins undergo misfolding and aggregation during this process, in a concentration-dependent fashion. Once formed, native and aggregated states are often kinetically trapped relative to each other. Hence, the early stages of protein life are absolutely critical for proper kinetic channeling to the folded state and for long-term solubility and function. This review summarizes current knowledge on protein folding/aggregation mechanisms in buffered solution and within the bacterial cell, highlighting early stages. Remarkably, teamwork between nascent chain, ribosome, trigger factor and Hsp70 molecular chaperones enables all proteins to overcome aggregation propensities and reach a long-lived bioactive state.

Enhanced nuclear-spin hyperpolarization of amino acids and proteins via reductive radical quenchers.

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as an effective tool for the hyperpolarization of aromatic amino acids in solution, either in isolation or within proteins. One factor limiting the maximum achievable signal-to-noise ratio in LC-photo-CIDNP is the progressive degradation of the target molecule and photosensitizer upon long-term optical irradiation. Fortunately, this effect does not cause spectral distortions but leads to a progressively smaller signal buildup upon long-term data-collection (e.g. 500 nM tryptophan on a 600 MHz spectrometer after ca. 200 scans). Given that it is generally desirable to minimize the extent of photodamage, we report that low-µM amounts of the reductive radical quenchers vitamin C (VC, i.e., ascorbic acid) or 2-mercaptoethylamine (MEA) enable LC-photo-CIDNP data to be acquired for significantly longer time than ever possible before. This approach increases the sensitivity of LC-photo-CIDNP by more than 100%, with larger enhancement factors achieved in experiments involving more transients. Our results are consistent with VC and MEA acting primarily by reducing transient free radicals of the NMR molecule of interest, thus attenuating the extent of photodamage. The benefits of this reductive radical-quencher approach are highlighted by the ability to collect long-term high-resolution 2D 1H-13C LC-photo-CIDNP data on a dilute sample of the drkN SH3 protein (5 µM).

Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis.

The relation between co- and post-translational protein folding and aggregation in the cell is poorly understood. Here, we employ a combination of fluorescence anisotropy decays in the frequency domain, fluorescence-detected solubility assays, and NMR spectroscopy to explore the role of the ribosome in protein folding within a biologically relevant context. First, we find that a primary function of the ribosome is to promote cotranslational nascent-protein solubility, thus supporting cotranslational folding even in the absence of molecular chaperones. Under these conditions, however, only a fraction of the soluble expressed protein is folded and freely tumbling in solution. Hence, the ribosome alone is insufficient to guarantee quantitative formation of the native state of the apomyoglobin (apoMb) model protein. Right after biosynthesis, nascent chains encoding apoMb emerge from the ribosomal exit tunnel and undergo a crucial irreversible post-translational kinetic partitioning between further folding and aggregation. Mutational analysis in combination with protein-expression kinetics and NMR show that nascent proteins can attain their native state only when the relative rates of soluble and insoluble product formation immediately upon release from the ribosome are tilted in favor of soluble species. Finally, the outcome of the above immediately post-translational kinetic partitioning is much more sensitive to amino acid sequence perturbations than the native fold, which is rather mutation-insensitive. Hence, kinetic channeling of nascent-protein conformation upon release from the ribosome may be a major determinant of evolutionary pressure.