Solution Nuclear Magnetic Resonance Research Articles

Cross-correlation effects in the solution NMR spectra of near-equivalent spin-1/2 pairs.

The nuclear magnetic resonance (NMR) spectra of spin-1/2 pairs contain four peaks, with two inner peaks much stronger than the outer peaks in the near-equivalence regime. We have observed that the strong inner peaks have significantly different linewidths when measurements were performed on a 13C2-labelled triyne derivative. This linewidth difference may be attributed to strong cross-correlation effects. We develop the theory of cross-correlated relaxation in the case of near-equivalent homonuclear spin-1/2 pairs, in the case of a molecule exhibiting strongly anisotropic rotational diffusion. Good agreement is found with the experimental NMR lineshapes.

Unusual Correlation between the Apparent Amorphous Solubility of a Drug and Solubilizer Concentration Revealed by NMR Analysis.

Herein, we investigated the effect of the solubilizers, cetyltrimethylammonium bromide (CTAB) and amino methacrylate copolymer (Eudragit E PO, EUD-E), on the apparent amorphous solubility of ketoprofen (KTP) and free KTP concentrations in an aqueous phase when a KTP-rich phase was generated by liquid-liquid phase separation. Quantitative analysis by solution nuclear magnetic resonance (NMR) revealed that the apparent amorphous solubility of KTP increased with increasing EUD-E concentrations by the solubilization of KTP into the EUD-E micelles; this was reminiscent of the improvement in the apparent crystalline solubility of KTP observed when EUD-E was added. In contrast, the apparent amorphous solubility of KTP decreased with increasing CTAB concentrations, although the solubilizing ability of CTAB was stronger than that of EUD-E when the KTP-rich phase was absent. NMR analysis revealed that CTAB was distributed into the KTP-rich phase to a relatively large extent. This resulted in a significant reduction of the chemical potential of KTP in the KTP-rich phase in the CTAB solution. Thus, the maximum free KTP concentration in the aqueous phase was reduced more significantly in the CTAB solution than in the EUD-E solution. Moreover, the solubilization effect of KTP by the CTAB micelles in the aqueous phase was drastically diminished due to the distribution of CTAB into the KTP-rich phase. As a result, the apparent amorphous solubility of KTP reached a minimum at a CTAB concentration of 200 μg/mL. A further increase in the CTAB concentration resulted in an improvement in the apparent amorphous solubility of KTP due to the solubilization effect of CTAB remaining in the aqueous phase. The present study highlights the impact of solubilizer selection on the apparent amorphous solubility and attainable supersaturation of the drug, which should be considered during the development of supersaturating formulations to obtain preferable oral absorption.

Molecular Mechanism of Antimicrobial Excipient-Induced Aggregation in Parenteral Formulations of Peptide Therapeutics.

Antimicrobial preservatives are used as functional excipients in multidose formulations of biological therapeutics to destroy or inhibit the growth of microbial contaminants, which may be introduced by repeatedly administering doses. Antimicrobial agents can also induce the biophysical instability of proteins and peptides, which presents a challenge in optimizing the drug product formulation. Elucidating the structural basis for aggregation aids in understanding the underlying mechanism and can offer valuable knowledge and rationale for designing drug substances and drug products; however, this remains largely unexplored due to the lack of high-resolution characterization. In this work, we utilize solution nuclear magnetic resonance (NMR) as an advanced biophysical tool to study an acylated 31-residue peptide, acyl-peptide A, and its interaction with commonly used antimicrobial agents, benzyl alcohol and m-cresol. Our results suggest that acyl-peptide A forms soluble octamers in the aqueous solution, which tumble slowly due to an increased molecular weight as measured by diffusion ordered spectroscopy and 1H relaxation measurement. The addition of benzyl alcohol does not induce aggregation of acyl-peptide A and has no chemical shift perturbation in 1H-1H NOESY spectra, suggesting no detectable interaction with the peptide. In contrast, the addition of 1% (w/v) m-cresol results in insoluble aggregates composed of 25% (w/w) peptides after a 24-hour incubation at room temperature as quantified by 1H NMR. Interestingly, 1H-13C heteronuclear single-quantum coherence and 1H-1H total correlation experiment spectroscopy have identified m-cresol and peptide interactions at specific residues, including Met, Lys, Glu, and Gln, suggesting that there may be a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with m-cresol driving this phenomenon. These site-specific interactions have promoted the formation of higher-order oligomerization such as dimers and trimers of octamers, eventually resulting in insoluble aggregates. Our study has elucidated a structural basis of m-cresol-induced self-association that can inform the optimized design of drug substances and products. Moreover, it has demonstrated solution NMR as a high-resolution tool to investigate the structure and dynamics of biological drug products and provide an understanding of excipient-induced peptide and protein aggregation.

Multilayer protein corona on gold nanorod surface: First evidence of soft corona protein-protein interactions using solution NMR spectroscopy

The nanoparticle surfaces interact readily with biomolecules, including proteins forming a corona. The protein layer that directly interacts with the nanoparticle surface is usually explored. Non-covalent interactions of proteins in the second layer have not been elucidated. We report for the first-time multilayer protein-protein interactions in ubiquitin corona on gold nanorod surface. The conformational dynamics of the protein, ubiquitin, on the surface of gold nanorods were probed at the molecular level using two-dimensional solution nuclear magnetic resonance spectroscopy. The interaction of Ubiquitin with gold nanorods was characterized by UV–visible spectroscopy, dynamic light scattering, and zeta-potential measurements. The residues of Ubiquitin that show significant perturbations fall into two categories: (a) the residues that directly interact with the surface of gold nanorods forming hard corona (primary interaction) and (b) the residues that are forming soft corona (secondary interaction). The formation of hard corona on the surface of the nanorod takes place even at a low concentration of the protein. The protein does not undergo any changes in its secondary or tertiary structure. As the protein concentration is increased, the hard corona, which is off mutually interconvertible multiple conformers, undergoes conformational reorientation to maximize its interaction with the nanorod surface. The study reveals electrostatically driven weak protein-nanorod interactions. Further, the study reveals two different processes, one involving the direct interaction with nanorods and the other involving protein corona formation in the protein-nanomaterial system. The study, for the first time, explores multilayer protein corona on a nanoparticle surface and provides new insights into the mechanisms involving protein aggregation in the presence of the nanoparticles.

Theoretical study of keto-enol tautomerism in 7-epi-clusianone by quantum chemical calculations of NMR chemical shifts.

Plants from the Garcinia genus have been used worldwide due to their therapeutic properties. Among the various metabolites isolated from this genus, 7-epi-clusianone, a tetraprenylated benzophenone, stands out for its wide range of identified biological activities. This benzophenone can exist in five tautomeric forms, although the benzene-d6 and chloroform-d3 solution nuclear magnetic resonance (NMR) spectra revealed only two tautomeric forms (B and C) in equilibrium, with concentration ratio depending on the solvent in which the spectrum was obtained. Calculated energy values suggested that tautomeric forms B and E would be prevalent in benzene-d6 solution, in contrast to the experimental data. Considering this conflicting result, we employed the statistical DP4 + method based on 13C and 1H NMR chemical shift calculations, in the gas phase and in benzene-d6 solution, to confirm that the B and C tautomeric forms of 7-epi-clusianone are the most prevalent in the experimental conditions.

Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex.

Dynamic solution nuclear magnetic resonance (NMR) spectroscopy is the typical method of characterizing the dynamic rearrangements of atoms within the coordination sphere for transition metal polyhydride complexes. Line shape fitting of the dynamic NMR spectra can lead to estimates for the activation parameters of the dynamic rearrangement processes. A combination of dynamic 31P-{1H} NMR spectroscopy of metal-bound phosphorus atoms with dynamic 1H-{31P} NMR spectroscopy of hydride ligands may identify hydride ligand rearrangements that occur in conjunction with a phosphorus atom rearrangement. For molecules that exhibit such a coupled pair of rearrangements, dynamic NMR spectroscopy can be used to test theoretical models for the ligand rearrangements. Dynamic 1H-{31P} NMR spectroscopy and line shape fitting can also identify the presence of an exchange process that moves a specific hydride ligand beyond the metal's inner coordination sphere through a proton exchange with a solvent molecule such as adventitious water. The preparation of a new compound, ReH5(PPh3)2(sec-butyl amine), that exemplifies multiple dynamic rearrangement processes is presented along with line shape fitting of dynamic NMR spectra of the complex. Line shape fitting results can be analyzed by the Eyring equation to estimate the activation parameters for the identified dynamic processes.

Multimetallic Permethylpentalene Hydride Complexes.

The synthesis and characterization of group 4 permethylpentalene (Pn* = C8Me6) hydride complexes are explored; in all cases, multimetallic hydride clusters were obtained. Group 4 lithium metal hydride clusters were obtained when reacting the metal dihalides with hydride transfer reagents such as LiAlH4, and these species featured an unusual hexagonal bipyramidal structural motif. Only the zirconium analogue was found to undergo hydride exchange in the presence of deuterium. In contrast, a trimetallic titanium hydride cluster was isolated on reaction of the titanium dialkyl with hydrogen. This diamagnetic, mixed valence species was characterized in the solid state, as well as by solution electron paramagnetic resonance and nuclear magnetic resonance spectroscopy. The structure was further probed and corroborated by density functional theory calculations, which illustrated the formation of a metal-cluster bonding orbital responsible for the diamagnetism of the complex. These permethylpentalene hydride complexes have divergent structural motifs and reactivity in comparison with related classical cyclopentadienyl analogues.

Targeting Pathogenic DNA and RNA Repeats: A Conceptual Therapeutic Way for Repeat Expansion Diseases

Expansions of short tandem repeats (STRs) in the human genome cause nearly 50 neurodegenerative diseases, which are mostly inheritable, nonpreventable and incurable, posing as a huge threat to human health. Non-B DNAs formed by STRs are thought to be structural intermediates that can cause repeat expansions. The subsequent transcripts harboring expanded RNA repeats can further induce cellular toxicity through forming specific structures. Direct targeting of these pathogenic DNA and RNA repeats has emerged as a new potential therapeutic strategy to cure repeat expansion diseases. In this conceptual review, we first introduce the roles of DNA and RNA structures in the genetic instabilities and pathomechanisms of repeat expansion diseases, then describe structural features of DNA and RNA repeats with a focus on the tertiary structures determined by X-ray crystallography and solution nuclear magnetic resonance spectroscopy, and finally discuss recent progress and perspectives of developing chemical tools that target pathogenic DNA and RNA repeats for curing repeat expansion diseases.

Conformational dynamics in GPCR signaling by NMR

G-protein-coupled receptors (GPCRs) mediate a wide range of cellular responses to various ligands or stimuli, and are the most important drug targets associated with human diseases. While major advances in GPCR structural biology have greatly deepened our understanding of its activation mechanism, the highly complex changes in the structural dynamics of GPCRs during activation remain underdetermined and their links to physiological functions largely unknown. Solution nuclear magnetic resonance (NMR) spectroscopy is an essential technique that allows the characterization of protein structural dynamics at atomic level, and has been applied in the studies of GPCR structural-function relationship in the past decade. Herein, we summarize a few specific studies in which solution NMR methods were employed and provided novel insights into questions difficult to be addressed by other methods.

Probing the Li Metal Solid Electrolyte Interphase Using a Stable Nitroxide Radical

The solid electrolyte interphase (SEI) on a Li metal anode is important in protecting the Li from further oxidation. Previous work has demonstrated that the SEI formed in a lithium-air battery (LAB) can be tailored depending on the gas species present. Oxygen helps create an oxide-rich SEI and promotes uniform Li metal deposition as opposed to dendritic Li which are formed when only argon is present. In order to compare the protective quality of the SEI formed under O2 or under Ar, we used TEMPO, (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl, as a probe. First, we studied the possible reduction products of TEMPO. We found that TEMPO initially reduces to TEMPO-, then undergoes further reductive degradation such as ring opening or formation of amines. Next, we exposed a Li metal electrode with SEI formed either under O2 or Ar to an electrolyte solution containing TEMPO. We characterized the ability of the SEI to prevent TEMPO reduction using operando Electron Paramagnetic Resonance (EPR) spectroscopy as well as ex situ solid-state and solution Nuclear Magnetic Resonance (NMR) spectroscopy. We show that the concentration of TEMPO in the electrolyte decreases more slowly and fewer TEMPO reduction products are formed when the Li metal has an oxide-rich SEI. This suggests that the oxide-rich SEI is better at protecting the Li metal from oxidation and that TEMPO is a suitable probe for SEI quality.

Investigating Transport through Separator Membranes in Aqueous Organic Redox Flow Batteries Using NMR Spectroscopy

Electricity and heat generation contribute 25% of greenhouse gas emissions globally.1 Redox flow batteries (RFBs) are prospective devices for long-duration energy storage, which is required to integrate more renewable energy sources onto the electricity grid.2 RFBs have a modular design with decoupled power and energy ratings, allowing them to be scaled to suit grid-level energy storage requirements. The development of aqueous organic redox flow batteries (AORFBs), such as quinone-based systems, is gaining momentum because they are potentially cheaper, safer and more sustainable than vanadium-based RFBs.3-5 However, the crossover of redox-active species through the separator membrane can lead to irreversible capacity fade, limiting their lifetime and economic viability.6 It has previously been demonstrated that in situ spectroscopic techniques are powerful tools for determining reaction mechanisms in redox flow batteries.5,7 Here, we explore how solution-state in situ nuclear magnetic resonance (NMR) spectroscopy and solid-state NMR spectroscopy can be used to study the crossover of electrolytes in AORFBs. We demonstrate that in situ solution NMR spectroscopy can be used to characterise transport in operating AORFBs with high temporal resolution and minimal system disturbance. This method can therefore be applied to investigate how crossover is governed by structure-property relationships and the charging protocols used. Furthermore, polymer-electrolyte interactions within the membrane can be probed using complementary solid-state NMR studies. Together, these fundamental studies will ultimately advance our understanding of electrolyte crossover, so that improved separator membranes can be developed.

NMR spectroscopy, excited states and relevance to problems in cell biology - transient pre-nucleation tetramerization of huntingtin and insights into Huntington's disease.

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for analyzing three-dimensional structure and dynamics of macromolecules at atomic resolution. Recent advances have exploited the unique properties of NMR in exchanging systems to detect, characterize and visualize excited sparsely populated states of biological macromolecules and their complexes, which are only transient. These states are invisible to conventional biophysical techniques, and play a key role in many processes, including molecular recognition, protein folding, enzyme catalysis, assembly and fibril formation. All the NMR techniques make use of exchange between sparsely populated NMR-invisible and highly populated NMR-visible states to transfer a magnetization property from the invisible state to the visible one where it can be easily detected and quantified. There are three classes of NMR experiments that rely on differences in distance, chemical shift or transverse relaxation (molecular mass) between the NMR-visible and -invisible species. Here, I illustrate the application of these methods to unravel the complex mechanism of sub-millisecond pre-nucleation oligomerization of the N-terminal region of huntingtin, encoded by exon-1 of the huntingtin gene, where CAG expansion leads to Huntington's disease, a fatal autosomal-dominant neurodegenerative condition. I also discuss how inhibition of tetramerization blocks the much slower (by many orders of magnitude) process of fibril formation.

Energy Landscape of the Sugar Conformation Controls the Sol-to-Gel Transition in Self-Assembled Bola Glycolipid Hydrogels

Self-assembled fibrillar network hydrogels and organogels are commonly obtained through a crystallization process as fibers upon being induced by external stimuli such as temperature or pH. The gel-to-sol-to-gel transition is generally readily reversible, and the rate of change of the stimulus determines the fiber homogeneity and eventual elastic properties of the gels. However, recent work shows that in some specific cases, fibrillation occurs for a given molecular conformation and the sol-to-gel transition depends on the relative energetic stability of one conformation over the other and not on the rate of change of the stimuli. We observe such a phenomenon on a class of bolaform glycolipids, sophorosides, similar to the well-known sophorolipid biosurfactants but composed of two symmetric sophorose units. A combination of oscillatory rheology, small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy, and in situ rheology coupled with SAXS using synchrotron radiation shows that below 14 °C, twisted nanofibers are the thermodynamic phase. Between 14 and about 33 °C, nanofibers coexist with micelles and a strong hydrogel forms, the sol-to-gel transition being readily reversible in this temperature range. However, above the annealing temperature of about 40 °C, the micelle morphology becomes kinetically trapped for hours, even upon cooling, whichever the rate, to 4 °C. A combination of solution and solid-state nuclear magnetic resonance spectroscopy studies suggests two different conformations of the 1″, 1′, and 2′ carbon stereocenters of sophorose, precisely at the β(1,2) glycosidic bond, for which several combinations of the dihedral angles are known to provide at least three energetic minima of comparable magnitude, with each corresponding to a given sophorose conformation.

Coordination of Palladium(II) and Platinum(II) Complexes to One Vacant Site in an α-Keggin-Type Polyoxotungstate.

A palladium-platinum bimetal complex containing a mono-lacunary α-Keggin-type polyoxotungstate is demonstrated. The bimetallic complex Cs2.5{PdII(bpy)}0.25[α-PW11O39{PdII(bpy)}{cis-PtII(NH3)2}]·12H2O·2CH3CN (bpy = 2,2'-bipyridine) (PdPt) was synthesized by the reaction of Cs4.9K0.1[α-PW11O39{cis-PtII(NH3)2}]·10H2O (Pt1) with PdII(bpy)Cl2 in aqueous solution. The compound PdPt was characterized via elemental analysis, thermogravimetric/differential thermal analysis, and Fourier transform infrared, ultraviolet-visible, cold-spray ionization mass, and solution (1H, 13C, and 31P) nuclear magnetic resonance spectroscopies. The coordination of {cis-PtII(NH3)2} and {PdII(bpy)} moieties to the mono-vacant site of [PW11O39]7- resulted in an asymmetric, monomeric compound. A unique exchange reaction between the counter cation [PdII(bpy)]2+ and {cis-PtII(NH3)2} moiety in [α-PW11O39{PdII(bpy)}{cis-PtII(NH3)2}]3- in a dimethylsulfoxide solution was also observed.

Difluorophosphoric Acid Generation and Crossover Reactions in LiNixCoyMnzO2 Cathodes Operating at High Voltage

Cycling lithiated metal oxides to high potential (>4.5 V vs Li) is of significant interest for the next generation of lithium ion batteries as this significantly increases the capacity and energy of cells. However, cells cycled to high potential suffer from rapid capacity fade due to a combination of thickening of the anode solid electrolyte interphase (SEI) and impedance growth on the cathode. While transition metal catalysed degradation of the anode SEI has been widely proposed as a primary source of capacity loss, a related acid induced degradation of the anode SEI is proposed. A systematic investigation of LiNi0.5Co0.2Mn0.3O2, and LiNi0.8Co0.1Mn0.1O2 cathodes cycled to 4.2 and 4.6 V has been conducted and the oxidative generation of the strong acid difluorophosphoric acid (F2PO2H) has been quantified by solution Nuclear Magnetic Resonance (NMR) spectroscopy. Ex-situ surface analysis of the electrodes with X-ray Photo Electron spectroscopy (XPS) suggests that the generation of F2PO2H correlates with a thickening of the anode SEI and an increase in the fluorophosphate content of the SEI. Changes to the LiNi0.8Co0.1Mn0.1O2 surface for cells cycled to 4.6 V are also consistent with the generation of acidic species. There is good correlation between the concentration F2PO2H, anode SEI degradation and the capacity loss of the cells.

Solution nuclear magnetic resonance spectroscopy of bacterial outer membrane proteins in natively excreted vesicles using engineered Escherichia coli.

Gaining structural information on membrane proteins in their native lipid environment is a long‐standing challenge in molecular biology. Instead, it is common to employ membrane mimetics, which has been shown to affect protein structure, dynamics, and function severely. Here, we describe the incorporation of a bacterial outer membrane protein (OmpW) into natively excreted membrane vesicles for solution nuclear magnetic resonance (NMR) spectroscopy using a mutant Escherichia coli strain with a high outer membrane vesicle (OMV) production rate. We collected NMR spectra from both vesicles containing overexpressed OmpW and vesicles from a control strain to account for the presence of physiologically relevant outer membrane proteins in vesicles and observed distinct resonance signals from OmpW. Due to the increased production of OMVs and the use of non‐uniform sampling techniques we were able to obtain high‐resolution 2D (HSQC) and 3D (HNCO) NMR spectra of our target protein inside its native lipid environment. While this workflow is not yet sufficient to achieve in situ structure determination, our results pave the way for further research on vesicle‐based solution NMR spectroscopy.

A Specific pSer/Thr-Pro Motif Generates Interdomain Communication Bifurcations of Two Modes of Pin1 in Solution Nuclear Magnetic Resonance.

Peptides mediate the interdomain communication of Pin1 (peptidyl-prolyl cis-trans isomerase) and can regulate its conformation and biochemical functions, providing an idea for drug design using Pin1. Two template peptide sequences have been widely used in the extended or compact state of Pin1 (Cdc25C, E-Q-P-L-pT-P-V-T-D-L; Pintide, W-F-Y-pS-P-R). The way in which specific pSer/Thr-Pro peptides regulate interdomain communication to achieve the opposite state is not clear. In this study, we subdivided the sequence composition of eight types of modified peptides and investigated the interaction with Pin1 by solution nuclear magnetic resonance and molecular dynamics. Demonstrating sequence dependence on the pSer-Pro or pThr-Pro motif and different residues in anchoring the WW domain, the Pin peptide (Pintide, PintideT, Pin25C, and Pin25CT) transmits this concentration accumulation to the PPIase domain, thus exhibiting two anchoring tendencies. However, the Cdc peptide (Cdc25C, Cdc25CS, Cdctide, and CdctideS) has a low binding energy that makes it difficult for the conformation to reach a steady state. In addition, Pin1 is influenced by both compact and extended states, regulated precisely by the sequence as well as by threonine or serine. These results provide new insight into the interdomain communication of Pin1 via pSer/Thr-Pro peptide binding.

Solution structure of c-FLIP death effector domains

The formation of death-inducing signaling complex (DISC) and death effector domain (DED) filament initiates extrinsic apoptosis. Recruitment and activation of procaspase-8 at the DISC are regulated by c-FLIP. The interaction between c-FLIP and procaspase-8 is mediated by their tandem DEDs (tDED). However, the structure of c-FLIPtDED and how c-FLIP interferes with procaspase-8 activation at the DISC remain elusive. Here, we solved the monomeric structure of c-FLIPtDED (F114G) at near physiological pH by solution nuclear magnetic resonance (NMR). Structural superimposition reveals c-FLIPtDED (F114G) adopts a structural topology similar to that of procaspase-8tDED. Our results provide a structural basis for understanding how c-FLIP interacts with procaspase-8 and the molecular mechanisms of c-FLIP in regulating cell death.

Formylation of Recombinant Spider Silk in Formic Acid and Wet Spinning Studied Using Nuclear Magnetic Resonance and Infrared Spectroscopies.

We reported wet spinning of recombinant spider silk protein (RSSP) and formylation of RSSP in formic acid (FA). First, FA was selected as the spinning solvent and the detailed spinning condition was determined. Next, the mechanical property was compared between the RSSP fiber spun after allowing the spinning solution dissolved in FA to stand for 2 days and the fiber spun immediately after being dissolved in FA for 4 h. The tensile strength of the former fiber was lower than the strength of the latter fiber. This difference can be explained by the difference in the degree of formylation as follows. FA is a known formylating agent, although most researchers who prepared silk fiber by wet spinning with FA have not pointed out about formylation. The formylation of the Ser OH group was confirmed by 13C solution nuclear magnetic resonance (NMR), and the time course of formylation of the RSSP film prepared from the FA solution was tracked by Fourier transform infrared spectroscopy. The 13C solid-state NMR spectra were also compared between two kinds of the formylated RSSP fibers and indicated that the packing state was tighter for the latter fiber than the former one, which could explain higher tensile strength of the latter fiber in the dry state. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RSSP sample decomposed gradually with storage time in FA and the decomposition has begun partly even at 2 h after dissolution in FA. The decomposition by formylation seems to have no significant effect on the backbone structure of the RSSP fiber, although the packing of the fiber becomes loose as a whole. Finally, preliminary trial of deformylation of the formylated RSSP fiber was performed.

Transient RNA Interactions Leave a Covalent Imprint on a Viral Capsid Protein.

The hepatitis B virus (HBV) is the leading cause of persistent liver infections. Its DNA-based genome is synthesized through reverse transcription of an RNA template inside the assembled capsid shell. In addition to the structured assembly domain, the capsid protein harbors a C-terminal extension that mediates both the enclosure of RNA during capsid assembly and the nuclear entry of the capsid during infection. The arginine-rich motifs within this extension, though common to many viruses, have largely escaped atomic-scale investigation. Here, we leverage solution and solid-state nuclear magnetic resonance spectroscopy at ambient and cryogenic temperatures, under dynamic nuclear polarization signal enhancement, to investigate the organization of the genome within the capsid. Transient interactions with phosphate groups of the RNA backbone confine the arginine-rich motifs to the interior capsid space. While no secondary structure is induced in the C-terminal extension, interactions with RNA counteract the formation of a disulfide bond, which covalently tethers this peptide arm onto the inner capsid surface. Electrostatic and covalent contributions thus compete in the spatial regulation of capsid architecture. This disulfide switch represents a coupling mechanism between the structured assembly domain of the capsid and the enclosed nucleic acids. In particular, it enables the redox-dependent regulation of the exposure of the C-terminal extension on the capsid surface, which is required for nuclear uptake of the capsid. Phylogenetic analysis of capsid proteins from hepadnaviruses points toward a function of this switch in the persistence of HBV infections.