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.

Solution-state NMR typically requires 100 μM to 1 mM samples. This limitation prevents applications to mass-limited and aggregationprone 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 H 1 -C 13 and side-chain resonances were enhanced, enabling secondary and tertiary structure analysis of proteins with remarkable spectral editing, via the 13 C 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. hyperpolarization | photo-CIDNP | NMR | proteins | amino acids N MR is an atomic-resolution noninvasive method to probe molecular structure and dynamics. This technique is, however, inherently insensitive due to the unfavorable distribution of nuclear spin states at the near-ambient temperature used in most applications. Methods implemented over the years to overcome the low sensitivity of NMR in liquids include the use of high applied magnetic fields, data acquisition in the time domain followed by Fourier transform, fast data collection schemes, and cryogenic probes (1-3). More recently, nuclear-spin hyperpolarization including Overhauser dynamic nuclear polarization (4, 5), optical pumping (6-9), parahydrogen-induced polarization (10, 11), signal amplification by reversible exchange (12), and dissolution dynamic nuclear polarization (D-DNP) (13-17) have displayed significant potential (3).Despite the above technological advances, typical liquid-state NMR experiments employing hyperpolarization still require very expensive instrumentation; harsh hyperpolarization conditions; long polarization times; and last but not least, ≥50-100 μM sample concentration. In addition, NMR data collection of dilute biomolecules in physiologically relevant milieux is often unfeasible. While some of the above challenges may in principle be overcome by concentrating NMR samples and employing probes accommodating small sample volumes, this process is often unfeasible due to limited amounts of available material or to undesirable aggregation. In summary, there is a compelling need to further enhance the sensitivity of solution-state NMR spectroscopy.Photochemically induced dynamic nuclear polarization (photo-CIDNP) is a spin-selective technique involving the transient generation of radical pairs (Fig. 1A). This methodology has been traditionally employed to gauge macromolecular solventexposure (18)(19)(20). More recently, phot...

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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 nascentprotein 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.