Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel.

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1-140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63-94 residues. Longer RNCs (apoHmp1-189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.

Mapping Protein-Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome-Nascent Globin Complex.

Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein-ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.

LC-Photo-CIDNP hyperpolarization of biomolecules bearing a quasi-isolated spin pair: Magnetic-Field dependence via a rapid-shuttling device.

Liquid-state low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an emerging technology tailored to enhance the sensitivity of NMR spectroscopy via LED- or laser-mediated optical irradiation. LC-photo-CIDNP is particularly useful to detect solvent-exposed aromatic residues (Trp, Tyr), either in isolation or within polypeptides and proteins. This study investigates the magnetic-field dependence of the LC-photo-CIDNP of Trp-α-13C-ß,ß,2,4,5,6,7-d7, a Trp isotopolog bearing a quasi-isolated 1Hα-13Cαspin pair (QISP). We employed a new rapid-shuttling side-illumination field-cycling device that enables ultra-fast (90-120 ms) vertical movements of NMR samples within the bore of a superconducting magnet. Thus, LC-photo-CIDNP hyperpolarization occurs at low field, while hyperpolarized signals are detected at high field (700 MHz). Resonance lineshapes were excellent, and the effect of several fields (1.18-7.08 T range) on hyperpolarization efficiency could be readily explored. Remarkably, unprecedented LC-photo-CIDNP enhancements ε ≅ 1,200 were obtained at 50 MHz (1.18 T), suggesting exciting avenues to hypersensitive LED-enhanced NMR in liquids at low field.

Magnetic-Field Dependence of LC-Photo-CIDNP in the Presence of Target Molecules Carrying a Quasi-Isolated Spin Pair.

NMR spectroscopy is well known for its superb resolution, especially at high applied magnetic field. However, the sensitivity of this technique is very low. Liquid-state low-concentration photo-chemically-induced dynamic nuclear polarization (LC-photo-CIDNP) is a promising emerging methodology capable of enhancing NMR sensitivity in solution. LC-photo-CIDNP works well on solvent-exposed Trp and Tyr residues, either in isolation or within proteins. This study explores the magnetic-field dependence of the LC-photo-CIDNP experienced by two tryptophan isotopologs in solution upon in situ LED-mediated optical irradiation. Out of the two uniformly 13C,15N-labeled Trp (Trp-U-13C,15N) and Trp-α-13C-ß,ß,2,4,5,6,7-d7 species employed here, only the latter bears a quasi-isolated 1Hα-13Cα spin pair. Computer simulations of the predicted polarization due to geminate recombination of both species display a roughly bell-shaped field dependence. However, while Trp-U-13C,15N is predicted to show a maximum at ca. 500 MHz (11.7 T) and a fairly weak field dependence, Trp-α-13C-ß,ß,2,4,5,6,7-d7 is expected to display a much sharper field dependence accompanied by a dramatic polarization increase at lower field (ca. 200 MHz, 4.7 T). Experimental LC-photo-CIDNP studies on both Trp isotopologs at 1µM concentration, performed at selected fields, are consistent with the theoretical predictions. In summary, this study highlights the prominent field-dependence of LC-photo-CIDNP enhancements (ε) experienced by Trp isotopologs bearing a quasi-isolated spin pair.

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.

Spin Hyperpolarization in Modern Magnetic Resonance.

Magnetic resonance techniques are successfully utilized in a broad range of scientific disciplines and in various practical applications, with medical magnetic resonance imaging being the most widely known example. Currently, both fundamental and applied magnetic resonance are enjoying a major boost owing to the rapidly developing field of spin hyperpolarization. Hyperpolarization techniques are able to enhance signal intensities in magnetic resonance by several orders of magnitude, and thus to largely overcome its major disadvantage of relatively low sensitivity. This provides new impetus for existing applications of magnetic resonance and opens the gates to exciting new possibilities. In this review, we provide a unified picture of the many methods and techniques that fall under the umbrella term "hyperpolarization" but are currently seldom perceived as integral parts of the same field. Specifically, before delving into the individual techniques, we provide a detailed analysis of the underlying principles of spin hyperpolarization. We attempt to uncover and classify the origins of hyperpolarization, to establish its sources and the specific mechanisms that enable the flow of polarization from a source to the target spins. We then give a more detailed analysis of individual hyperpolarization techniques: the mechanisms by which they work, fundamental and technical requirements, characteristic applications, unresolved issues, and possible future directions. We are seeing a continuous growth of activity in the field of spin hyperpolarization, and we expect the field to flourish as new and improved hyperpolarization techniques are implemented. Some key areas for development are in prolonging polarization lifetimes, making hyperpolarization techniques more generally applicable to chemical/biological systems, reducing the technical and equipment requirements, and creating more efficient excitation and detection schemes. We hope this review will facilitate the sharing of knowledge between subfields within the broad topic of hyperpolarization, to help overcome existing challenges in magnetic resonance and enable novel applications.

Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome.

Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding-site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client-protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome-wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258 Escherichia coli (E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent-exposed than other nonpolar sites, in protein native states. Our findings show that the majority of E. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70-bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70-chaperone-binding traits have evolved to favor Hsp70-assisted protein folding devoid of aggregation.

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.

Selective Isotope Labeling and LC-Photo-CIDNP Enable NMR Spectroscopy at Low-Nanomolar Concentration.

NMR spectroscopy is a powerful tool to investigate molecular structure and dynamics. The poor sensitivity of this technique, however, limits its ability to tackle questions requiring dilute samples. Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an optically enhanced NMR technology capable of addressing the above challenge by increasing the detection limit of aromatic amino acids in solution up to 1000-fold, either in isolation or within proteins. Here, we show that the absence of NMR-active nuclei close to a magnetically active site of interest (e.g., the structurally diagnostic 1Hα-13Cα pair of amino acids) is expected to significantly increase LC-photo-CIDNP hyperpolarization. Then, we exploit the spin-diluted tryptophan isotopolog Trp-α-13C-ß,ß,2,4,5,6,7-d7 and take advantage of the above prediction to experimentally achieve a ca 4-fold enhancement in NMR sensitivity over regular LC-photo-CIDNP. This advance enables the rapid (within seconds) detection of 20 nM concentrations or the molecule of interest, corresponding to a remarkable 3 ng detection limit. Finally, the above Trp isotopolog is amenable to incorporation within proteins and is readily detectable at a 1 µM concentration in complex cell-like media, including Escherichia coli cell-free extracts.

An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel.

The influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins.

This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57-83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.

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).

Net Charge and Nonpolar Content Guide the Identification of Folded and Prion Proteins.

The degree of hydrophobicity and net charge per residue are physical properties that enable the discrimination of folded from intrinsically disordered proteins (IDPs) solely on the basis of amino acid sequence. Here, we improve upon the existing classification of proteins and IDPs based on the parameters mentioned above by adopting the scale of nonpolar content of Rose et al. and by taking amino acid side-chain acidity and basicity into account. The resulting algorithm, denoted here as net charge nonpolar or NECNOP, enables the facile prediction of the folded and disordered status of proteins under physiologically relevant conditions with >95% accuracy, based on amino-acid sequence alone. The NECNOP approach displays a much-enhanced performance for proteins with >140 residues, suggesting that small proteins are more likely to have irregular charge and hydrophobicity features. NECNOP analysis of the entire Escherichia coli proteome identifies specific net charge and nonpolar regions peculiar to soluble, integral membrane, and non-integral membrane proteins. Surprisingly, protein net charge and hydrophobicity are found to converge to specific values as chain length increases, across the E. coli proteome. In addition, NECNOP plots enable the straightforward identification of protein sequences corresponding to prion proteins and promise to serve as a powerful predictive tool for the design of large proteins. In summary, NECNOP plots are a straightforward approach that improves our understanding of the relation between the amino acid sequence and three-dimensional structure of proteins as a function of molecular mass.

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.

Modular control of l-tryptophan isotopic substitution via an efficient biosynthetic cascade.

Isotopologs are powerful tools for investigating biological systems. We report a biosynthetic-cascade synthesis of Trp isotopologs starting from indole, glycine, and formaldehyde using the enzymes l-threonine aldolase and an engineered ß-subunit of tryptophan synthase. This modular route to Trp isotopologs is simple and inexpensive, enabling facile access to these compounds.

KLR-70: A Novel Cationic Inhibitor of the Bacterial Hsp70 Chaperone.

The heat-shock factor Hsp70 and other molecular chaperones play a central role in nascent protein folding. Elucidating the task performed by individual chaperones within the complex cellular milieu, however, has been challenging. One strategy for addressing this goal has been to monitor protein biogenesis in the absence and presence of inhibitors of a specific chaperone, followed by analysis of folding outcomes under both conditions. In this way, the role of the chaperone of interest can be discerned. However, development of chaperone inhibitors, including well-known proline-rich antimicrobial peptides, has been fraught with undesirable side effects, including decreased protein expression yields. Here, we introduce KLR-70, a rationally designed cationic inhibitor of the Escherichia coli Hsp70 chaperone (also known as DnaK). KLR-70 is a 14-amino acid peptide bearing naturally occurring residues and engineered to interact with the DnaK substrate-binding domain. The interaction of KLR-70 with DnaK is enantioselective and is characterized by high affinity in a buffered solution. Importantly, KLR-70 does not significantly interact with the DnaJ and GroEL/ES chaperones, and it does not alter nascent protein biosynthesis yields across a wide concentration range. Some attenuation of the anti-DnaK activity of KLR-70, however, has been observed in the complex E. coli cell-free environment. Interestingly, the d enantiomer D-KLR-70, unlike its all-L KLR-70 counterpart, does not bind the DnaK and DnaJ chaperones, yet it strongly inhibits translation. This outcome suggests that the two enantiomers (KLR-70 and D-KLR-70) may serve as orthogonal inhibitors of chaperone binding and translation. In summary, KLR-70 is a novel chaperone inhibitor with high affinity and selectivity for bacterial Hsp70 and with considerable potential to help in parsing out the role of Hsp70 in nascent protein folding.

High-Resolution Diffusion Measurements of Proteins by NMR under Near-Physiological Conditions.

Measuring the translational diffusion of proteins under physiological conditions can be very informative, especially when multiple diffusing species can be distinguished. Diffusion NMR or diffusion-ordered spectroscopy (DOSY) is widely used to study molecular diffusion, where protons are used as probes, which can be further edited by the proton-attached heteronuclei to provide additional resolution. For example, the combination of the backbone amide protons (1HN) to measure diffusion with the well-resolved 1H/15N correlations has afforded high-resolution DOSY experiments. However, significant amide-water proton exchange at physiological temperature and pH can affect the accuracy of diffusion data or cause complete loss of DOSY signals. Although aliphatic protons do not exchange with water protons, and thus are potential probes to measure diffusion rates, 1H/13C correlations are often in spectral overlap or masked by the water signal, which hampers the use of these correlations. In this report, a method was developed that separates the nuclei used for diffusion (α protons, 1Hα) and those used for detection (1H/15N and 13C'/15N correlations). This approach enables high-resolution diffusion measurements of polypeptides in a mixture of biomolecules, thereby providing a powerful tool to investigate coexisting species under physiologically relevant conditions.

Improved sensitivity of laser-enhanced 1Hα-13Cα-correlation via suppression of Cα-C' scalar-coupling evolution.

Low-concentration photochemically induced dynamic polarization (LC-photo-CIDNP) enables the spectroscopic analysis of biomolecules containing the amino acids Trp and Tyr at sub-micromolar concentration in solution. Typical LC-photo-CIDNP pulse sequences involving 1H-13C correlation, however, perform well in the case of aromatic resonances but display a relatively poor signal-to-noise ratio for 13Cα and 13Cß resonances. Here, we develop a novel pulse sequence denoted as 13C perturbation-recovered selective-pulse photo-CINDP enhanced reverse INEPT, or 13C PRESPRINT, tailored to the LC-photo-CIDNP analysis of 1H-13Cα pairs. Our method, which is based on full suppression of 1-bond Cα-C' scalar-coupling evolution during the constant-time delay, results into a sensitivity improvement by a factor of 2. The enhanced performance of this pulse sequence enabled us to improve the analysis of LC-photo-CIDNP laser-power dependence at very low (200 nM) sample concentration. An improved theoretical model, developed to quantitatively describe this laser-power dependence, shows excellent agreement with our 13C PRESPRINT experimental data.

Kinetic trapping in protein folding.

The founding principles of protein folding introduced by Christian Anfinsen, together with the numerous mechanistic investigations that followed, assume that protein folding is a thermodynamically controlled process. On the other hand, this review underscores the fact that thermodynamic control is far from being the norm in protein folding, as long as one considers an extended chemical-potential landscape encompassing aggregates, in addition to native, unfolded and intermediate states. Here, we highlight the key role of kinetic trapping of the protein native state relative to unfolded, intermediate and, most importantly, aggregated states. We propose that kinetic trapping serves an important role in biology by protecting the bioactive states of a large number of proteins from deleterious aggregation. In the event that undesired aggregates were somehow formed, specialized intracellular disaggregation machines have evolved to convert any aberrant populations back to the native state, thus restoring a fully bioactive and aggregation-protected protein cohort.