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.

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.

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.

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.

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.

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.

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.

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.

Fast-pulsing LED-enhanced NMR: A convenient and inexpensive approach to increase NMR sensitivity.

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as a powerful technology for the detection of aromatic amino acids and proteins in solution in the low-micromolar to nanomolar concentration range. LC-photo-CIDNP is typically carried out in the presence of high-power lasers, which are costly and maintenance-heavy. Here, we show that LC-photo-CIDNP can be performed with light-emitting diodes (LEDs), which are inexpensive and much less cumbersome than lasers, laser diodes, flash lamps, or other light sources. When nuclear magnetic resonance (NMR) sample concentration is within the low-micromolar to nanomolar range, as in LC-photo-CIDNP, replacement of lasers with LEDs leads to no losses in sensitivity. We also investigate the effect of optical-fiber thickness and compare excitation rate constants of an Ar ion laser (488 nm) and a 466 nm LED, taking LED emission bandwidths into account. In addition, importantly, we develop a novel pulse sequence (13C RASPRINT) to perform ultrarapid LC-photo-CIDNP data collection. Remarkably, 13C RASPRINT leads to 4-fold savings in data collection time. The latter advance relies on the fact that photo-CID nuclear hyperpolarization does not suffer from the longitudinal-relaxation recovery requirements of conventional NMR. Finally, we combine both the above improvements, resulting in facile and rapid (≈16 s-2.5 min) collection of 1 and 2D NMR data on aromatic amino acids and proteins in solution at nanomolar to low micromolar concentration.

Heterogeneous binding of the SH3 client protein to the DnaK molecular chaperone.

The molecular chaperone heat shock protein 70 (Hsp70) plays a vital role in cellular processes, including protein folding and assembly, and helps prevent aggregation under physiological and stress-related conditions. Although the structural changes undergone by full-length client proteins upon interaction with DnaK (i.e., Escherichia coli Hsp70) are fundamental to understand chaperone-mediated protein folding, these changes are still largely unexplored. Here, we show that multiple conformations of the SRC homology 3 domain (SH3) client protein interact with the ADP-bound form of the DnaK chaperone. Chaperone-bound SH3 is largely unstructured yet distinct from the unfolded state in the absence of DnaK. The bound client protein shares a highly flexible N terminus and multiple slowly interconverting conformations in different parts of the sequence. In all, there is significant structural and dynamical heterogeneity in the DnaK-bound client protein, revealing that proteins may undergo some conformational sampling while chaperone-bound. This result is important because it shows that the surface of the Hsp70 chaperone provides an aggregation-free environment able to support part of the search for the native state.

Charge segregation and low hydrophobicity are key features of ribosomal proteins from different organisms.

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.

Burial of nonpolar surface area and thermodynamic stabilization of globins as a function of chain elongation.

Proteins are biosynthesized from N to C terminus before they depart from the ribosome and reach their bioactive state in the cell. At present, very little is known about the evolution of conformation and the free energy of the nascent protein with chain elongation. These parameters critically affect the extent of folding during ribosome-assisted biosynthesis. Here, we address the impact of vectorial amino acid addition on the burial of nonpolar surface area and on the free energy of native-like structure formation in the absence of the ribosomal machinery. We focus on computational predictions on proteins bearing the globin fold, which is known to encompass the 3/3, 2/2, and archaeal subclasses. We find that the burial of nonpolar surface increases progressively with chain elongation, leading to native-like conformations upon addition of the last C-terminal residues, corresponding to incorporation of the last two helices. Additionally, the predicted folding entropy for generating native-like structures becomes less unfavorable at nearly complete chain lengths, suggesting a link between the late burial of nonpolar surface and water release. Finally, the predicted folding free energy takes a progressive favorable dip toward more negative values, as the chain gets longer. These results suggest that thermodynamic stabilization of the native structure of newly synthesized globins during translation in the cell is significantly enhanced as the chain elongates. This is especially true upon departure of the last C-terminal residues from the ribosomal tunnel, which hosts ca., 30-40 amino acids. Hence, we propose that release from the ribosome is a crucial step in the life of single-domain proteins in the cell.

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.

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.

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.

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.

High-resolution conformation and backbone dynamics of a soluble aggregate of apomyoglobin119.

The structure and dynamics of soluble misfolded aggregates are poorly understood, despite their importance in protein science and disease. Water-soluble self-associated species that do not become insoluble over time are invaluable tools for high-resolution conformational studies aimed at dissecting the determinants of self-association. Here, we characterize the soluble model aggregate apomyoglobin(119) (apoMb(119)), generated upon truncating the residues corresponding to the C-terminal helix of sperm whale apomyoglobin. The secondary structure and backbone dynamics of apoMb(119), determined by multidimensional NMR at pH 6.0, reveal the presence of an N-terminal slow-tumbling core and a highly disordered flexible C-terminus displaying residual helicity and large-amplitude backbone motions on the picosecond-to-nanosecond timescale. The backbone of the apoMb(119) aggregate assumes progressively increased mobility as residues get further removed from the nonpolar core and closer to the more hydrophilic C-terminal end. This structural motif establishes a useful paradigm for the topology of soluble misfolded protein aggregates in aqueous solution in the absence of stabilizing additives. The partially helical and flexible C-terminus of apoMb(119)'s aggregate is in interesting contrast with the amyloid-related globulomers, which display dangling ends rich in ß-strand. Finally, we investigate how a molecular chaperone, the substrate-binding domain of DnaK, interferes with apoMb(119)'s aggregation.

1H-Detected 13C photo-CIDNP as a sensitivity enhancement tool in solution NMR.

NMR is a powerful yet intrinsically insensitive technique. The applicability of NMR to chemical and biological systems would be substantially extended by new approaches going beyond current signal-to-noise capabilities. Here, we exploit the large enhancements arising from (13)C photochemically induced dynamic nuclear polarization ((13)C photo-CIDNP) in solution to improve biomolecular NMR sensitivity in the context of heteronuclear correlation spectroscopy. The (13)C-PRINT pulse sequence presented here involves an initial (13)C nuclear spin polarization via photo-CIDNP followed by conversion to anti-phase coherence and transfer to (1)H for detection. We observe substantial enhancements, up to ≫200-fold, relative to the dark (laser off) experiment. Resonances of both side-chain and backbone CH pairs are enhanced for the three aromatic residues Trp, His, and Tyr, the σ(32) peptide, and the drkN SH3 protein. The sensitivity of this experiment, defined as signal-to-noise per square root of unit time (S/N)(t), is unprecedented in the NMR polarization enhancement literature dealing with polypeptides in solution. Up to a 16-fold larger (S/N)(t) than for the (1)H-(13)C SE-HSQC reference sequence is achieved, for the σ(32) peptide. Data collection time is reduced up to 256-fold, highlighting the advantages of (1)H-detected (13)C photo-CIDNP in solution NMR.