RESUMEN
The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.
Asunto(s)
Amiloide/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Alineación de SecuenciaRESUMEN
While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C'-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.
Asunto(s)
Frío , Proteínas , Temperatura , Espectroscopía de Resonancia Magnética , Conformación Proteica , Proteínas/química , Resonancia Magnética Nuclear BiomolecularRESUMEN
Molecular clusters can function as nanoscale atoms/superatoms, assembling into superatomic solids, a new class of solid-state materials with designable properties through modifications on superatoms. To explore possibilities on diversifying building blocks, here we thoroughly studied one representative superatom, Co6 Se8 (PEt3 )6 . We probed its structural, electronic, and magnetic properties and revealed its detailed electronic structure as valence electrons delocalize over inorganic [Co6 Se8 ] core while ligands function as an insulated shell. 59 Co SSNMR measurements on the core and 31 P, 13 C on the ligands show that the neutral Co6 Se8 (PEt3 )6 is diamagnetic and symmetric, with all ligands magnetically equivalent. Quantum computations cross-validate NMR results and reveal degenerate delocalized HOMO orbitals, indicating aromaticity. Ligand substitution keeps the inorganic core nearly intact. After losing one electron, the unpaired electron in [Co6 Se8 (PEt3 )6 ]+1 is delocalized, causing paramagnetism and a delocalized electron spin. Notably, this feature of electron/spin delocalization over a large cluster is attractive for special single-electron devices.
RESUMEN
Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dipéptidos/química , Agregado de Proteínas , Secuencia de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Precipitación Química , Dipéptidos/fisiología , Humanos , Concentración de Iones de Hidrógeno , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Dominios y Motivos de Interacción de Proteínas/fisiología , Solventes/química , Solventes/farmacologíaRESUMEN
Magic angle spinning NMR rotating frame relaxation measurements provide a unique experimental window into biomolecules dynamics, as is illustrated by numerous recent applications. We discuss experimental strategies for this class of experiments, with a particular focus on systems where motion-driven modulation of the chemical shift interaction is the main mechanism for relaxation. We also explore and describe common strategies for interpreting the data sets to extract motion time scale, activation energy, and angle or order parameters from rotating frame relaxation data. Using model free analysis and numerical simulations, including time domain treatment, we explore conditions under which it is possible to obtain accurate and precise information about the time scales of motions. Overall, with rapid technical advances in solid state NMR, there is a bright future for this class of studies.
Asunto(s)
Imagen por Resonancia Magnética , Biopolímeros , Espectroscopía de Resonancia Magnética , Movimiento (Física) , Resonancia Magnética Nuclear BiomolecularRESUMEN
Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.
Asunto(s)
Proteínas Bacterianas/metabolismo , Canales de Potasio/metabolismo , Proteínas Bacterianas/genética , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Mutación , Potasio/metabolismo , Canales de Potasio/genética , Conformación ProteicaRESUMEN
TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/ultraestructura , Antígeno Intracelular 1 de las Células T/ultraestructura , Proteínas Intrínsecamente Desordenadas/metabolismo , Espectroscopía de Resonancia Magnética , Micelas , Microscopía Electrónica , Modelos Moleculares , Pliegue de Proteína , Multimerización de Proteína , Motivos de Unión al ARN , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Antígeno Intracelular 1 de las Células T/metabolismoRESUMEN
We report here an iterative synthesis of long helical perylene diimide (hPDI[n]) nanoribbons with a length up to 16 fused benzene rings. These contorted, ladder-type conjugated, and atomically precise nanoribbons show great potential as organic fast-charging and long-lifetime battery cathodes. By tuning the length of the hPDI[n] oligomers, we can simultaneously modulate the electrical conductivity and ionic diffusivity of the material. The length of the ladders adjusts both the conjugation for electron transport and the contortion for lithium-ion transport. The longest oligomer, hPDI[6], when fabricated as the cathode in lithium batteries, features both high electrical conductivity and high ionic diffusivity. This electrode material exhibits a high power density and can be charged in less than 1 min to 66% of its maximum capacity. Remarkably, this material also has exceptional cycling stability and can operate for up to 10,000 charging-discharging cycles without any appreciable capacity decay. The design principles described here chart a clear path for organic battery electrodes that are sustainable, fast-charging, and long lasting.
RESUMEN
Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.
Asunto(s)
Regulación Alostérica , Modelos Moleculares , Proteínas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Espectroscopía de Resonancia Magnética , Mutación , Canales de Potasio/química , Canales de Potasio/metabolismo , Conformación Proteica , Proteínas/genética , Relación Estructura-ActividadRESUMEN
As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.
RESUMEN
The power of chemical shift anisotropy (CSA) measurements for probing structure and dynamics of molecules has been long recognized. NMR pulse sequences that allow measurement of CSA values in an indirect dimension of a protein correlation spectrum have been employed for aliphatic groups, but for practical reasons, carbonyl functional groups have been little studied, despite the fact that carbonyls are expected to give particularly varied and informative CSA values. Specifically, the wide spectral widths of carbonyl tensors make their measurements difficult with typically attainable spectrometer settings. We present here an extended family of experiments that enable the recovery of static CSA lineshapes in an indirect dimension of magic angle spinning (MAS) solid-state NMR experiments, except for various real valued scaling factors. The experiment is suitable for uniformly labeled material, at moderate MAS rates (10 kHz-30 kHz) and at higher magnetic fields (ν0H > 600 MHz). Specifically, the experiments are based on pulse sequence elements from a previous commonly used pulse sequence for CSA measurement, recoupling of chemical shift anisotropy (ROCSA), while modification of scaling factors is achieved by interspersing different blocks of C-elements of the same Cnn 1 cycle. Using experimental conditions similar to the parent ROCSA sequence, a CSA scaling factor between 0 and 0.272 can be obtained, thus allowing a useful practical range of possibilities in experimental conditions for measurement of larger CSA values. Using these blocks, it is also possible to make a constant-time CSA recoupling sequence. The effectiveness of this approach, fROCSA, is shown on model compounds 1-13C-Gly, U-13C,15N-l-His, and microcrystalline U-13C,15N-Ubiquitin.
Asunto(s)
Aminoácidos/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Algoritmos , Anisotropía , Isótopos de Carbono/análisis , Campos Magnéticos , Isótopos de Nitrógeno/análisis , Resonancia Magnética Nuclear Biomolecular/métodos , Ubiquitina/químicaRESUMEN
The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K+ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 Å from the K+ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Canales de Potasio/química , Potasio/química , Protones , Regulación Alostérica , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Membrana Dobles de Lípidos/metabolismo , Potasio/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismoRESUMEN
An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.
Asunto(s)
Bacteriófago Pf1/química , Resonancia Magnética Nuclear Biomolecular , Pseudomonas aeruginosa/virología , Bacteriófago Pf1/metabolismoRESUMEN
Magic angle spinning solid state NMR studies of biological macromolecules [1-3] have enabled exciting studies of membrane proteins [4,5], amyloid fibrils [6], viruses, and large macromolecular assemblies [7]. Dynamic nuclear polarization (DNP) provides a means to enhance detection sensitivity for NMR, particularly for solid state NMR, with many recent biological applications and considerable contemporary efforts towards elaboration and optimization of the DNP experiment. This review explores precedents and innovations in biological DNP experiments, especially highlighting novel chemical biology approaches to introduce the radicals that serve as a source of polarization in DNP experiments.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas/metabolismo , Animales , HumanosRESUMEN
It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.
Asunto(s)
Sitio Alostérico , Proteínas Bacterianas/química , Membrana Celular/química , Canales de Potasio/fisiología , Ácidos Carboxílicos/química , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Potasio/química , Canales de Potasio/química , Estructura Secundaria de Proteína , Proteínas/químicaRESUMEN
The bacterial chemoreceptor complex governs signal detection and the upstream elements of chemotactic behavior, but the detailed molecular mechanism is still unclear. We have assembled nativelike functional arrays of an aspartate receptor cytoplasmic fragment (CF) with its two cytoplasmic protein partners (CheA and CheW) for solid-state nuclear magnetic resonance (NMR) studies of structural changes involved in signaling. In this initial study of the uniformly (13)C- and (15)N-enriched CF in these >13.8 MDa size arrays, residue-type assignments are made for amino acids that together make up 90% of the protein. We demonstrate that homo- and heteronuclear two-dimensional spectra are consistent with structure-based chemical shift predictions: a number of major assignable correlations are consistent with the predominantly α-helical secondary structure, and minor correlations are consistent with the disordered C-terminal tail. Sub-parts per million line widths and spectral changes upon freezing of samples suggest these arrays are structurally homogeneous and sufficiently immobilized for efficient solid-state NMR.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Receptores de Superficie Celular/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de ProteínaRESUMEN
The interaction of lipids with subunit c from F1F0 ATP synthase is studied by biophysical methods. Subunit c from both Escherichia coli and Streptococcus pneumoniae interacts and copurifies with cardiolipin. Solid state NMR data on oligomeric rings of F0 show that the cardiolipin interacts with the c subunit in membrane bilayers. These studies offer strong support for the hypothesis that F0 has specific interactions with cardiolipin.
Asunto(s)
Cardiolipinas/química , Espectroscopía de Resonancia Magnética/métodos , ATPasas de Translocación de Protón Mitocondriales/química , Subunidades de ProteínaRESUMEN
We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.
Asunto(s)
Gramicidina/metabolismo , Proteínas de la Membrana/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Espectroscopía de Resonancia por Spin del Electrón , Gramicidina/química , Membrana Dobles de Lípidos , Proteínas de la Membrana/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Dominios y Motivos de Interacción de Proteínas , Marcadores de SpinRESUMEN
The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K(+) titrations, we detect a local change at E71 that is coupled to channel collapse at low K(+). To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K(+) and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pK(a) (> 7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA-E71A does not inactivate and establish the ambient K(+) level as a means to populate the inactivated state of KcsA in a controlled way.
Asunto(s)
Proteínas Bacterianas/química , Canales de Potasio/química , Streptomyces lividans/metabolismo , Anisotropía , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Iones , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , Conformación Molecular , Mutación , Potasio/química , Canales de Potasio/genética , Conformación Proteica , Estructura Terciaria de Proteína , Protones , Sodio/químicaRESUMEN
High resolution two- and three-dimensional heteronuclear correlation spectroscopy ((1)H-(13)C, (1)H-(15)N, and (1)H-(13)C-(13)C HETCOR) has provided a detailed characterization of the internal and external hydration water of the Pf1 virion. This long and slender virion (2000 nm × 7 nm) contains highly stretched DNA within a capsid of small protein subunits, each only 46 amino acid residues. HETCOR cross-peaks have been unambiguously assigned to 25 amino acids, including most external residues 1-21 as well as residues 39-40 and 43-46 deep inside the virion. In addition, the deoxyribose rings of the DNA near the virion axis are in contact with water. The sets of cross-peaks to the DNA and to all 25 amino acid residues were from the same hydration water (1)H resonance; some of the assigned residues do not have exchangeable side-chain protons. A mapping of the contacts onto structural models indicates the presence of water "tunnels" through a highly hydrophobic region of the capsid. The present results significantly extend and modify results from a lower resolution study, and yield a comprehensive hydration surface map of Pf1. In addition, the internal water could be distinguished from external hydration water by means of paramagnetic relaxation enhancement. The internal water population may serve as a conveniently localized magnetization reservoir for structural studies.