Most natively folded proteins exhibit a unique spatial structure, which undergoes functional motions ranging from picoseconds to seconds, governed by a hierarchically ordered, funnel-shaped free energy landscape. Intrinsically disordered proteins (IDPs) lack such a stable native structure, but undergo fast interconversions between many different structures. Accordingly, the underlying free energy landscape is assumed to be rather shallow and unstructured. However, although IDPs represent nearly one-third of the human proteome, their structural dynamics on timescales slower than nanoseconds remain largely elusive. Here we reveal the structural dynamics of the prototypical IDP p53-TAD, also known as the "guardian of the genome", by combining high-power relaxation dispersion nuclear magnetic resonance spectroscopy with large-scale molecular dynamics simulations. We found a complex hierarchy of structural dynamics on timescales covering over seven orders of magnitude, ranging from fast nanoseconds backbone reorientations, via sub-microsecond helix-formation dynamics involving many structural sub-states and transition times, to transient tertiary structure formation slower than 25 microseconds. These rich structural dynamics are unexpectedly similar to the timescale hierarchy of natively folded proteins, which may be key to the ability of p53-TAD - and possibly of other IDPs - to bind many different partners by folding into different structures.

Sequence-specific backbone resonance assignment remains the most critical and time-limiting step in biomolecular NMR investigations of protein structure, dynamics, and interactions. Despite major advances in spectrometer hardware, cryogenic probes, and accelerated acquisition schemes, the assignment problem continues to pose serious challenges for proteins that are unstable in solution, intrinsically disordered, α-helical, or rich in repetitive sequence motifs. In such systems, severe chemical shift degeneracy—particularly of backbone amide 1H and 13C resonances—often renders conventional triple-resonance strategies inefficient, ambiguous, or impractical within the available sample lifetime. Traditional backbone assignment protocols rely on multiple complementary three-dimensional experiments (e.g., HNCA/HN(CO)CA, HNCACB/CBCA(CO)NH, HNCO/HN(CA)CO), which collectively demand long experimental times and extensive manual analysis. Although robust, these approaches are inherently low-throughput and frequently fail in the presence of high backbone degeneracy or conformational heterogeneity. These limitations motivated the development of alternative assignment paradigms that minimize experimental redundancy while maximizing internal consistency and robustness.In this review, we present a comprehensive and unified account of the HNN and HN(C)N suite of NMR experiments, tracing their evolution from the original conceptual framework to modern tunable, reduced-dimensionality, and automated implementations. We emphasize the underlying principles that make these experiments uniquely powerful for rapid backbone assignment and backbone structure determination across a wide range of protein systems. By consolidating methodological developments, application strategies, and automation tools, this review aims to serve both as a reference and as a practical guide for researchers seeking efficient solutions to the backbone assignment problem in contemporary biomolecular NMR. While extraction of reliable NOE-derived distance restraints remains one of the most time-consuming steps in high-resolution protein structure determination, it is critically dependent on accurate and unambiguous backbone resonance assignment. In this context, the HNN and HN(C)N suite of experiments provide a robust and efficient framework for rapid backbone assignment, thereby enabling and accelerating downstream structural analysis.

A new protocol based on the 3D-hNCAnH experiment for rapid, unambiguous backbone resonance assignment and estimation of structural information by intensity quantification of the peaks is described here. Along F1-F3 plane at F2(13Cαi) of the 3D-hNCAnH spectrum, each i to i + 1 sequential connectivity (i.e., HiNi → Hi + 1Ni + 1) is confirmed by two inter-residue sequential correlation peaks: HiNi + 1 and Hi + 1Ni. This allows unambiguous and direct identification of sequential correlations in HSQC peaks, without the need for extensive searching in different planes of 3D spectra. Further, a protocol utilizing the ratio of the intensities of the diagonal and cross peaks along F3(1H) dimension centered at self F2(13Cαi) and sequential F2(13Cαi-1) chemical shifts taken from F2-F3 plane at F1(15Ni) of the 3D-hNCAnH spectrum for estimating one and two bond N-Cα J-coupling constants, respectively, is described. The reliability of this approach is demonstrated using doubly labelled ubiquitin protein, wherein the coupling constants that are measured by the method described here are compared with previously measured values (BMRB Nos.: 15907 and 16582). The application of the approach to other proteins is demonstrated using doubly labelled human SUMO and Ca2+ bound M-crystallin proteins.

Intrinsically disordered proteins (IDPs) exhibit pronounced structural dynamics, which is crucial for their functional versatility. Yet their dynamics slower than nanoseconds remain largely elusive. We combined high-power relaxation dispersion nuclear magnetic resonance spectroscopy with molecular dynamics simulations to characterize these kinetics and the underlying structural interconversions of a prototypical IDP, the N-terminal transactivation domain of the tumor suppressor p53 (p53-TAD). We find a complex hierarchy of structural dynamics on timescales covering over seven orders of magnitude, ranging from fast nanoseconds backbone re-orientations, via sub-microsecond helix-formation dynamics involving many structural sub-states and transition times, to transient tertiary structure formation slower than 25 microseconds. These rich structural dynamics of p53-TAD, and likely those of other IDPs, parallel the timescale hierarchy of the conformational dynamics of folded proteins. One-Sentence summary A hierarchical energy landscape governs kinetics and structural dynamics of the disordered p53 transactivation domain. ### Competing Interest Statement The authors have declared no competing interest. Alexander von Humboldt Foundation, https://ror.org/012kf4317 Deutsche Forschungsgemeinschaft, EXC 2067/1-390729940 Korea Basic Science Institute, C539200, C523400, C526112 Max Planck Society, https://ror.org/01hhn8329 Max Planck Computing and Data Facility, https://ror.org/03e21z229

Chemotaxis Y (CheY), upon metal binding, displays a drastic alteration in its structure and stability. This premise prompted us to study the effect of crowding on the two conformationally distinct states of the same test protein. A comparative analysis on the structure and thermal stability in the presence and absence of the macromolecular crowder, ficoll, and its monomeric unit, sucrose, revealed a contrasting effect of ficoll on the apo and holo forms. In the presence of ficoll while the thermal stability (Tm) of the apo form is enhanced, the thermal stability of the holo form is reduced. The selective lowering of Tm for the holo form in the combined presence of ficoll and sucrose and not in sucrose alone suggests that the contrasting effect is due to the macromolecular nature of ficoll. Since metal-protein interaction remains unperturbed in the presence of ficoll and Mg2+ sequestration is ruled out in a systematic manner the alternative possibility for the exclusive reduction in the thermal stability of the holo form is the ficoll-induced modulation of the relative population of apo and holo forms of CheY.

A novel three-component strategy has been developed for the synthesis of iminosugars in good to excellent yields. This is the first report on the Mannich type addition of cyclic 1,3-diketones to aza-acetal derived from hydroxy-γ-lactone and arylamine to produce a novel series of aza-sugars with high selectivity.

y-Hydroxyolefin derived from proline undergoes a smooth ene cyclization with aldehydes to produce the 7-oxa-1-azaspiro[4.5]decane scaffolds in good yields. Whereas the condensation of y-hydroxyalkene with 2-hydroxybenzaldehyde affords the corresponding tetrahydro-2H-spiro[pyrano[3,2-c]chromene-3,20-pyrrolidine derivatives in excellent yields. This is the first report on the ene cyclization of aldehydes with y-hydroxyalkene derived from the a-amino acid, proline.CO 2022 Elsevier Ltd. All rights reserved.

The prevailing understanding of various aspects of biochemical processes, including folding, stability, intermolecular interactions, and the binding of metals, substrates, and inhibitors, is derived from studies carried out under dilute and homogeneous conditions devoid of a crowding-related environment. The effect of crowding-induced modulation on the structure and stability of native and magnesium-dependent Chemotaxis Y (CheY), a bacterial signaling protein, was probed in the presence and absence of poly(ethylene glycol) (PEG). A combined analysis from circular dichroism, intrinsic and extrinsic fluorescence, and tryptophan fluorescence lifetime changes indicates that PEG perturbs the structure but leaves the thermal stability largely unchanged. Intriguingly, while the stability of the protein is enhanced in the presence of magnesium under dilute buffer conditions, PEG-induced crowding leads to reduced thermal stability in the presence of magnesium. Nuclear magnetic resonance (NMR) chemical shift perturbations and resonance broadening for a subset of residues indicate that PEG interacts specifically with a subset of hydrophilic and hydrophobic residues found predominantly in α helices, β strands, and in the vicinity of the metal-binding region. Thus, PEG prompted conformational perturbation, presumably provides a different situation for magnesium interaction, thereby perturbing the magnesium-prompted stability. In summary, our results highlight the dominance of enthalpic contributions between PEG and CheY via both hydrophilic and hydrophobic interactions, which can subtly affect the conformation, modulating the metal-protein interaction and stability, implying that in the context of cellular situation, structure, stability, and magnesium binding thermodynamics of CheY may be different from those measured in dilute solution.

The Ugi four-centre three-component strategy has been developed for the synthesis of iminosugars in good yields under catalyst-free conditions. This method proceeds through a bicyclic N-acyl-iminium intermediate followed by the attack of isonitrile resulting in the formation of dihydroxypyroglutamic acid, which is a core moiety of many biologically important natural products

g-Hydroxyolefin derived from proline undergoes a smooth ene cyclization with aldehydes to produce the 7-oxa-1-azaspiro[4.5]decane scaffolds in good yields. Whereas the condensation of g-hydroxyalkene with 2-hydroxybenzaldehyde affords the corresponding tetrahydro-2H-spiro[pyrano[3,2-c]chromene-3,2'-pyrrolidine derivatives in excellent yields. This is the first report on the ene cyclization of aldehydes with g-hydroxyalkene derived from the a-amino acid, proline

The Ugi four-centre three-component strategy has been developed for the synthesis of iminosugars in good yields under catalyst-free conditions. This method proceeds through a bicyclic N-acyl-iminium intermediate followed by the attack of isonitrile resulting in the formation of dihydroxypyroglutamic acid, which is a core moiety of many biologically important natural products.(c) 2022 Elsevier Ltd. All rights reserved.

Unfolding followed by fibrillation of insulin even in the presence of various excipients grappled with restricted clinical application. Thus, there is an unmet need for better thermostable, nontoxic molecules to preserve bioactive insulin under varying physiochemical perturbations. In search of cross-amyloid inhibitors, prion-derived tetrapeptide library screening reveals a consensus V(X)YR motif for potential inhibition of insulin fibrillation. A tetrapeptide VYYR, isosequential to the β2-strand of prion, effectively suppresses heat- and storage-induced insulin fibrillation and maintains insulin in a thermostable bioactive form conferring adequate glycemic control in mouse models of diabetes and impedes insulin amyloidoma formation. Besides elucidating the critical insulin-IS1 interaction (R4 of IS1 to the N24 insulin B-chain) by nuclear magnetic resonance spectroscopy, we further demonstrated non-canonical dimer-mediated conformational trapping mechanism for insulin stabilization. In this study, structural characterization and preclinical validation introduce a class of tetrapeptide toward developing thermostable therapeutically relevant insulin formulations.

Molecular level insights on protein-ionic liquid (P-IL) interactions are beneficial for assessing protein stability, binding and dynamics. In the present work, interactions of ILs, namely, 1-butyl 3-methylimidazolium methyl sulfate (IL1), 1-butyl 3-methylimidazolium octyl sulfate (IL2) and 1-butyl 3-methylimidazolium chloride (IL3) with hen egg white lysozyme (HEWL) protein were investigated using solution-state nuclear magnetic resonance (NMR) spectroscopy. To ascertain the binding and dynamics from the perspective of both protein and IL, various ligand based NMR approaches such as selective and non-selective nuclear spin-relaxation (R1SEL and R1NS), saturation transfer difference (STD), difference of inversion recovery rate with and without target irradiation (DIRECTION), 35Cl line-shape and spin-relaxation, and protein back bone amide chemical shift perturbations (CSPs) from 1H-15N HSQC were utilized. Among the ILs investigated, IL2 experiences significant interaction relative to those of IL1 and IL3, as revealed by the combined R1SEL and R1NS analysis, which is further supported by STD NMR. CSP analyses of 1H-15N HSQC spectra of aqueous P-IL mixtures enabled to identify the potential binding sites of ILs with HEWL. Whereas, 15N longitudinal (R1) and transverse (R2) spin-relaxation rates and 15N{1H} heteronuclear nuclear Overhauser effect (hetNOE) data subjected to the model free analysis for IL2 yielded the rotational correlation times and order parameters of various residues of HEWL. Furthermore, the results could discern the nature of interactions between studied ILs and HEWL in terms of specific and non-specific interactions.

Deutsches Zentrum für Neurodegenerative E 37075 Göttingen, Germany. E-mail: mazw@ Max-Planck-Institut für Biophysikalische Ch Germany NMR & Structural Chemistry Division, CSIR Hyderabad, 500007, India Deutsches Zentrum für Neurodegenerative Allee 2, 53175 Bonn, Germany CAESAR Research Center, Bonn, MPI for Me 22607 Hamburg, Germany † Electronic supplementary informa 10.1039/c9sc00531e Cite this: Chem. Sci., 2019, 10, 6503

Liquid-liquid phase separation (LLPS) of proteins enables the formation of non-membrane-bound organelles in cells and is associated with cancer and neurodegeneration. Little is known however about the structure and dynamics of proteins in LLPS conditions, because of the polymorphic nature of liquid-like protein droplets. Using carbon-detected NMR experiments we here show that the conversion of the aggregation-prone repeat region of the Alzheimer's-related protein tau from the dispersed monomeric state to phase-separated liquid-like droplets involves tau's aggregation-prone hexapeptides and regulatory KXGS motifs. Droplet dissolution in presence of 1,6-hexanediol revealed that chemical shift perturbations in the hexapeptide motifs are temperature driven, while those in KXGS motifs report on phase separation. Residue-specific secondary structure analysis further indicated that tau's repeat region exists in extended conformation in the dispersed state and attains transient β-hairpin propensity upon LLPS. Taken together our work shows that NMR spectroscopy can provide high-resolution insights into LLPS-induced changes in intrinsically disordered proteins.

A wide array of aldehydes undergo smooth cross‐coupling with 5,6‐bis(2‐hydroxyethyl)‐2‐phenyl‐3a,4,7,7a‐tetrahydro‐1H‐isoindole‐1,3(2H)‐dione in the presence of 1.2 equiv. TMSOTf at –78 °C in dichloromethane to afford the corresponding hexahydro‐8a,4a‐(epoxyethano)pyrano[3,4‐f]isoindole‐1,3(2H,5H)‐dione derivatives in good yields with excellent diastereoselectivity. This is the first report on the stereoselective construction of oxa‐bridged tetracyclic frameworks through a bicyclization strategy.

The first asymmetric total synthesis of macrolactone monocillin VII and its C-10' epimer was achieved starting from a known chiral pure epoxide in 16 longest linear sequences. The present synthesis highlights the macrolactone formation involving an alkyne-dicobalt carbonyl complex under De Brabander's conditions followed by an unexpected regioselective hydration. The asymmetric total synthesis resulted in the revision of the configuration at C10' and reassignment of the absolute configuration of the natural product.

Molecular dynamics play a significant role in how molecules perform their function. A critical method that provides information on dynamics, at the atomic level, is NMR-based relaxation dispersion (RD) experiments. RD experiments have been utilized for understanding multiple biological processes occurring at micro-to-millisecond time, such as enzyme catalysis, molecular recognition, ligand binding and protein folding. Here, we applied the recently developed high-power RD concept to the Carr-Purcell-Meiboom-Gill sequence (extreme CPMG; E-CPMG) for the simultaneous detection of fast and slow dynamics. Using a fast folding protein, gpW, we have shown that previously inaccessible kinetics can be accessed with the improved precision and efficiency of the measurement by using this experiment.