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Closed-form expressions for the analysis of Dark state Exchange Saturation Transfer (DEST) NMR experiments, a powerful experimental tool for characterizing exchange processes involving the interaction of NMR visible species with very high molecular weight partners, is presented. Essentially identical exchange and relaxation parameters are derived from the analytical and numerical best fits of the DEST profiles obtained for a protein construct derived from huntingtin exon-1, comprising the N-terminal amphiphilic sequence followed by a seven-residue glutamine repeat, htt NT Q 7 , in the presence of small (SUV) and large (LUV) unilamellar lipid vesicles. The use of analytical expressions significantly speeds up the fitting of experimental DEST profiles to a two-state exchange model and simplifies the analysis of the DEST effects.

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a β-roll upon binding the ops -paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops -paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the β3/β4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15 N backbone chemical shift differences are associated with the β3/β4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH 2 groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing 15 N and 1 H components of NH 2 multiplets. Although even modest sensitivity gains in 2D NH 2 -TROSY correlation maps compared to their decoupled NH 2 -HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH 2 sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH 2 -TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH 2 positions of the L99A T4 lysozyme mutant - a model system for studies of the role of protein dynamics in ligand binding., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Double electron-electron resonance (DEER) EPR is a powerful tool in structural biology, providing distances between pairs of spin labels. When the sample consists of a mixture of oligomeric species (e.g., monomer and dimer), the question arises as to how to assign the peaks in the DEER-derived probability distance distribution to the individual species. Here, we propose incorporating an EPR longitudinal electron relaxation ( T 1 ) inversion recovery experiment within a DEER pulse sequence to resolve this problem. The apparent T 1 between dipolar coupled electron spins measured from the inversion recovery time (τ inv ) dependence of the peak intensities in the T 1 -edited DEER-derived probability P ( r ) distance distribution will be affected by the number of nitroxide labels attached to the biomolecule of interest, for example, two for a monomer and four for a dimer. We show that global fitting of all the T 1 -edited DEER echo curves, recorded over a range of τ inv values, permits the deconvolution of distances between spin labels originating from monomeric (longer T 1 ) and dimeric (shorter T 1 ) species. This is especially useful when the trapping of spin labels in different conformational states during freezing gives rise to complex P ( r ) distance distributions. The utility of this approach is demonstrated for two systems, the β 1 adrenergic receptor and a construct of the huntingtin exon-1 protein fused to the immunoglobulin domain of protein G, both of which exist in a monomer-dimer equilibrium.

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hht ex1 ) with a 35 polyglutamine repeat, htt ex1 Q 35 , are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of htt ex1 Q 35 aggregation, monitored by measuring cross-peak intensities in a series of 2D 1 H- 15 N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" htt ex1 Q 35 tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, htt ex1 Q 7 , lend confidence to the conclusions drawn from the fits to the htt ex1 Q 35 aggregation kinetics.

Pathogenic huntingtin exon-1 protein (htt ex1 ), characterized by an expanded polyglutamine tract located between the N-terminal amphiphilic region and a C-terminal polyproline-rich domain, forms fibrils that accumulate in neuronal inclusion bodies, and is associated with a fatal, autosomal dominant neurodegenerative condition known as Huntington's disease. Here a complete kinetic model is described for aggregation/fibril formation of a htt ex1 construct with a 35-residue polyglutamine repeat, htt ex1 Q 35 . Using exchange NMR spectroscopy, it is previously shown that the reversible formation of a sparsely-populated tetramer of the N-terminal amphiphilic domain of htt ex1 Q 35 , comprising a D 2 symmetric four-helix bundle, occurs on the microsecond time-scale and is a prerequisite for subsequent nucleation and fibril formation on a time scale that is many orders of magnitude slower (hours). Here a unified kinetic model of htt ex1 Q 35 aggregation is developed in which fast, reversible tetramerization is directly linked to slow irreversible fibril formation via conversion of pre-equilibrated tetrameric species to "active", chain elongation-capable nuclei by conformational re-arrangement with a finite, monomer-independent rate. The unified model permits global quantitative analysis of reversible tetramerization and irreversible fibril formation from a time series of 1 H- 15 N correlation spectra recorded during the course of htt ex1 Q 35 aggregation., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Advanced Science published by Wiley‐VCH GmbH.)

The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor-NIH biomolecular structure determination package. In combination with an improved treatment of selected non-bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters are tested using structure calculations on 30 proteins with restraints derived from nuclear magnetic resonance data. Using modern structure validation criteria, including several formally adopted by the Protein Data Bank, and a clear measure of structural accuracy, the results show superior performance relative to previous Xplor-NIH implementations. Additionally, the Xplor-NIH structures compare favorably against originally determined NMR models., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Longitudinal (Γ 1 ) and transverse (Γ 2 ) solvent paramagnetic relaxation enhancement (sPRE) yields field-dependent information in the form of spectral densities that provides unique information related to cosolute-protein interactions and electrostatics. A typical protein sPRE data set can only sample a few points on the spectral density curve, J (ω), within a narrow frequency window (500 MHz to ∼1 GHz). However, complex interactions and dynamics of paramagnetic cosolutes around a protein make it difficult to directly interpret the few experimentally accessible points of J (ω). In this paper, we show that it is possible to significantly extend the experimentally accessible frequency range (corresponding to a range from ∼270 MHz to 1.8 GHz) by acquiring a series of sPRE experiments at different temperatures. This approach is based on the scaling property of J (ω) originally proposed by Melchior and Fries for small molecules. Here, we demonstrate that the same scaling property also holds for geometrically far more complex systems such as proteins. Using the extended spectral densities derived from the scaling property as the reference dataset, we demonstrate that our previous approach that makes use of a non-Lorentzian Ansatz spectral density function to fit only J (0) and one to two J (ω) points allows one to obtain accurate values for the concentration-normalized equilibrium average of the electron-proton interspin separation ⟨ r -6 ⟩ norm and the correlation time τ C , which provide quantitative information on the energetics and timescale, respectively, of local cosolute-protein interactions. We also show that effective near-surface potentials, ϕ ENS , obtained from ⟨ r -6 ⟩ norm provide a reliable and quantitative measure of intermolecular interactions including electrostatics, while ϕ ENS values obtained from only Γ 1 or Γ 2 sPRE rates can have significant artifacts as a consequence of potential variations and changes in the diffusive properties of the cosolute around the protein surface. Finally, we discuss the experimental feasibility and limitations of extracting the high-frequency limit of J (ω) that is related to ⟨ r -8 ⟩ norm and report on the extremely local intermolecular potential.

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13 C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13 C- 1 H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964-73, 2004) supplemented with a CPMG train of refocusing 1 H pulses applied with constant frequency and synchronized with the 13 C CPMG pulse train. The optimal 1 H 'decoupling' scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1 H pulses. For small-to-medium sized proteins, the MQ 13 C CPMG experiment has the advantage over its single quantum (SQ) 13 C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13 C CPMG experiment eliminates complications in the interpretation of MQ 13 C- 1 H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1 H chemical shifts between ground and excited states. The MQ 13 C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The chaperone Hsp104, a member of the Hsp100/Clp family of translocases, prevents fibril formation of a variety of amyloidogenic peptides in a paradoxically substoichiometric manner. To understand the mechanism whereby Hsp104 inhibits fibril formation, we probed the interaction of Hsp104 with the Alzheimer's amyloid-β42 (Aβ42) peptide using a variety of biophysical techniques. Hsp104 is highly effective at suppressing the formation of Thioflavin T (ThT) reactive mature fibrils that are readily observed by atomic force (AFM) and electron (EM) microscopies. Quantitative kinetic analysis and global fitting was performed on serially recorded 1 H- 15 N correlation spectra to monitor the disappearance of Aβ42 monomers during the course of aggregation over a wide range of Hsp104 concentrations. Under the conditions employed (50 μM Aβ42 at 20 °C), Aβ42 aggregation occurs by a branching mechanism: an irreversible on-pathway leading to mature fibrils that entails primary and secondary nucleation and saturating elongation; and a reversible off-pathway to form nonfibrillar oligomers, unreactive to ThT and too large to be observed directly by NMR, but too small to be visualized by AFM or EM. Hsp104 binds reversibly with nanomolar affinity to sparsely populated Aβ42 nuclei present in nanomolar concentrations, generated by primary and secondary nucleation, thereby completely inhibiting on-pathway fibril formation at substoichiometric ratios of Hsp104 to Aβ42 monomers. Tight binding to sparsely populated nuclei likely constitutes a general mechanism for substoichiometric inhibition of fibrillization by a variety of chaperones. Hsp104 also impacts off-pathway oligomerization but to a much smaller degree initially reducing and then increasing the rate of off-pathway oligomerization.

The bacterial MinE and MinD division regulatory proteins form a standing wave enabling MinC, which binds MinD, to inhibit FtsZ polymerization everywhere except at the midcell, thereby assuring correct positioning of the cytokinetic septum and even distribution of contents to daughter cells. The MinE dimer undergoes major structural rearrangements between a resting six-stranded state present in the cytoplasm, a membrane-bound state, and a four-stranded active state bound to MinD on the membrane, but it is unclear which MinE motifs interact with the membrane in these different states. Using NMR, we probe the structure and global dynamics of MinE bound to disc-shaped lipid bicelles. In the bicelle-bound state, helix α1 no longer sits on top of the six-stranded β-sheet, losing any contact with the protein core, but interacts directly with the bicelle surface; the structure of the protein core remains unperturbed and also interacts with the bicelle surface via helix α2. Binding may involve a previously identified excited state of free MinE in which helix α1 is disordered, thereby allowing it to target the membrane surface. Helix α1 and the protein core undergo nanosecond rigid body motions of differing amplitudes in the plane of the bicelle surface. Global dynamics on the sub-millisecond time scale between a ground state and a sparsely populated excited state are also observed and may represent a very early intermediate on the transition path between the resting six-stranded and active four-stranded conformations. In summary, our results provide insights into MinE structural rearrangements important during bacterial cell division., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

G protein-coupled receptors (GPCR) activate numerous intracellular signaling pathways. The oligomerization properties of GPCRs, and hence their cellular functions, may be modulated by various components within the cell membrane (such as the presence of cholesterol). Modulation may occur directly via specific interaction with the GPCR or indirectly by affecting the physical properties of the membrane. Here, we use pulsed Q-band double electron-electron resonance (DEER) spectroscopy to probe distances between R1 nitroxide spin labels attached to Cys163 and Cys344 of the β 1 -adrenergic receptor (β 1 AR) in n -dodecyl-β-D-maltoside micelles upon titration with two soluble cholesterol analogs, cholesteryl hemisuccinate (CHS) and sodium cholate. The former, like cholesterol, inserts itself into the lipid membrane, parallel to the phospholipid chains; the latter is aligned parallel to the surface of membranes. Global quantitative analysis of DEER echo curves upon titration of spin-labeled β 1 AR with CHS and sodium cholate reveal the following: CHS binds specifically to the β 1 AR monomer at a site close to the Cys163-R1 spin label with an equilibrium dissociation constant [Formula: see text] ~1.4 ± 0.4 mM. While no direct binding of sodium cholate to the β 1 AR receptor was observed by DEER, sodium cholate induces specific β 1 AR dimerization ([Formula: see text] ~35 ± 6 mM and a Hill coefficient n  ~ 2.5 ± 0.4) with intersubunit contacts between transmembrane helices 1 and 2 and helix 8. Analysis of the DEER data obtained upon the addition of CHS to the β 1 AR dimer in the presence of excess cholate results in dimer dissociation with species occupancies as predicted from the individual K D values.

Degenerate spin-systems consisting of magnetically equivalent nuclear spins, such as a 1 H 3 spin-system in selectively 13 CH 3 -labeled proteins, present considerable challenges for the design of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments to characterize chemical exchange on the micro-to-millisecond time-scale. Several approaches have been previously proposed for the elimination of deleterious artifacts observed in methyl 1 H CPMG relaxation dispersion profiles obtained for ( 13 C) 1 H 3 groups. We describe an alternative, experimentally simple solution and design a "steady-state" methyl 1 H CPMG scheme, where 90° or acute-angle (<90°) 1 H radiofrequency pulses are applied after each CPMG echo in-phase with methyl 1 H magnetization, resulting in the establishment of a "steady-state" for effective rates of magnetization decay. A simple computational procedure for quantitative analysis of the "steady-state" CPMG relaxation dispersion profiles is developed. The "steady-state" CPMG methodology is applied to two protein systems where exchange between major and minor species occurs in different regimes on the chemical shift time-scale.

Solvent paramagnetic relaxation enhancement (sPRE) arising from nitroxide-based cosolutes has recently been used to provide an atomic view of cosolute-induced protein denaturation and to characterize residue-specific effective near-surface electrostatic potentials (ϕ ENS ). Here, we explore distinct properties of the sPRE arising from nitroxide-based cosolutes and provide new insights into the interpretation of the sPRE and sPRE-derived ϕ ENS . We show that: (a) the longitudinal sPRE rate Γ 1 is heavily dependent on spectrometer field and viscosity, while the transverse sPRE rate Γ 2 is much less so; (b) the spectral density J (0) is proportional to the inverse of the relative translational diffusion constant and is related to the quantity ⟨ r -4 ⟩ norm , a concentration-normalized equilibrium average of the electron-proton interspin separation; and (c) attractive intermolecular interactions result in a shortening of the residue-specific effective correlation time for the electron-proton vector. We discuss four different approaches for evaluating ϕ ENS based on Γ 2 , J (0), Γ 1 , or ⟨ r -6 ⟩ norm . The latter is evaluated from the magnetic field dependence of Γ 1 in conjunction with Γ 2 . Long-range interactions dominate J (0) and Γ 2 , while, at high magnetic fields, the contribution of short-range interactions becomes significant for J (ω) and hence Γ 1 ; the four ϕ ENS quantities enable one to probe both long- and short-range electrostatic interactions. The experimental ϕ ENS potentials were evaluated using three model protein systems, two folded (ubiquitin and native drkN SH3) and one intrinsically disordered (unfolded state of drkN SH3), in relation to theoretical ϕ ENS potentials calculated from atomic coordinates using the Poisson-Boltzmann theory with either a r -6 or r -4 dependence.

The global motions of ubiquitin, a model protein, on the surface of anisotropically tumbling 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-(1'-rac-glycerol) (POPG):1,2-dihexanoyl- sn -glycero-3-phosphocholine (DHPC) bicelles are described. The shapes of POPG:DHPC bicelles prepared with high molar ratios q of POPG to DHPC can be approximated by prolate ellipsoids, with the ratio of ellipsoid dimensions and dimensions themselves increasing with higher values of q . Adaptation of the nuclear magnetic resonance (NMR) relaxation-based approach that we previously developed for interactions of ubiquitin with spherical POPG liposomes (Ceccon, A. J. Am. Chem. Soc. 2016, 138, 5789-5792) allowed us to quantitatively analyze the variation in lifetime line broadening of NMR signals (Δ R 2 ) measured for ubiquitin in the presence of q = 2 POPG:DHPC bicelles and the associated transverse spin relaxation rates ( R 2,B ) of bicelle-bound ubiquitin. Ubiquitin, transiently bound to POPG:DHPC bicelles, undergoes internal rotation about an axis orthogonal to the surface of the bicelle and perpendicular to the principal axis of its rotational diffusion tensor on the low microsecond time scale (∼3 μs), while the rotation axis itself wobbles in a cone on a submicrosecond time scale (≤ 500 ns).

The N-terminal region of the huntingtin protein, encoded by exon-1 (htt ex1 ) and containing an expanded polyglutamine tract, forms fibrils that accumulate in neuronal inclusion bodies, resulting in Huntington's disease. We previously showed that reversible formation of a sparsely populated tetramer of the N-terminal amphiphilic domain, comprising a dimer of dimers in a four-helix bundle configuration, occurs on the microsecond timescale and is an essential prerequisite for subsequent nucleation and fibril formation that takes place orders of magnitude slower on a timescale of hours. For pathogenic htt ex1 , such as htt ex1 Q 35 with 35 glutamines, NMR signals decay too rapidly to permit measurement of time-intensive exchange-based experiments. Here, we show that quantitative analysis of both the kinetics and mechanism of prenucleation tetramerization and aggregation can be obtained simultaneously from a series of 1 H- 15 N band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC) correlation spectra. The equilibria and kinetics of tetramerization are derived from the time dependence of the 15 N chemical shifts and 1 H- 15 N cross-peak volume/intensity ratios, while the kinetics of irreversible fibril formation are afforded by the decay curves of 1 H- 15 N cross-peak intensities and volumes. Analysis of data on htt ex1 Q 35 over a series of concentrations ranging from 200 to 750 μM and containing variable (7 to 20%) amounts of the Met 7 O sulfoxide species, which does not tetramerize, shows that aggregation of native htt ex1 Q 35 proceeds via fourth-order primary nucleation, consistent with the critical role of prenucleation tetramerization, coupled with first-order secondary nucleation. The Met 7 O sulfoxide species does not nucleate but is still incorporated into fibrils by elongation.

Calcium-loaded calmodulin (CaM/4Ca 2+ ) comprises two domains that undergo rigid body reorientation from a predominantly extended conformation to a compact one upon binding target peptides. A recent replica-exchange molecular dynamics (MD) simulation on holo CaM/4Ca 2+ suggested the existence of distinct structural clusters (substates) along the path from extended to compact conformers in the absence of substrates. Here, we experimentally demonstrate the existence of CaM/4Ca 2+ substates trapped in local minima by three freezing/annealing regimes (slow, 40 s; intermediate, 1.5 s; fast, 0.5 ms) using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure interdomain distances between nitroxide spin-labels positioned at A17C and A128C in the N- and C-terminal domains, respectively. The DEER echo curves were directly fit to population-optimized P ( r ) pairwise distance distributions calculated from the coordinates of the MD clusters and compact crystal structure. DEER data on fully deuterated CaM/4Ca 2+ were acquired at multiple values of the second echo period (10-35 μs) and analyzed globally to eliminate instrumental and overfitting artifacts and ensure accurate populations, peak positions, and widths. The DEER data for all three freezing regimes are quantitatively accounted for within experimental error by 5-6 distinct conformers comprising a predominantly populated extended form (60-75%) and progressively more compact states whose populations decrease as the degree of compactness increases. The shortest interdomain separation is found in the compact crystal structure, which has an occupancy of 4-6%. Thus, CaM/4Ca 2+ samples high energy local minima comprising a few discrete substates of increasing compactness in a rugged energy landscape.

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Molecular chaperones are the guardians of the proteome inside the cell. Chaperones recognize and bind unfolded or misfolded substrates, thereby preventing further aggregation; promoting correct protein folding; and, in some instances, even disaggregating already formed aggregates. Chaperones perform their function by means of an array of weak protein-protein interactions that take place over a wide range of timescales and are therefore invisible to structural techniques dependent upon the availability of highly homogeneous samples. Nuclear magnetic resonance (NMR) spectroscopy, however, is ideally suited to study dynamic, rapidly interconverting conformational states and protein-protein interactions in solution, even if these involve a high-molecular-weight component. In this review, we give a brief overview of the principles used by chaperones to bind their client proteins and describe NMR methods that have emerged as valuable tools to probe chaperone-substrate and chaperone-chaperone interactions. We then focus on a few systems for which the application of these methods has greatly increased our understanding of the mechanisms underlying chaperone functions.

DNAJB6 is a prime example of an anti-aggregation chaperone that functions as an oligomer. DNAJB6 oligomers are dynamic and subunit exchange is critical for inhibiting client protein aggregation. The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces both chaperone self-oligomerization and anti-aggregation of client proteins, and has recently been linked to Parkinson's disease. Here, we show by NMR, including relaxation-based methods, that the T193A mutation has minimal effects on the structure of the β-stranded CTD but increases the population and rate of formation of a partially folded state. The results can be rationalized in terms of β-strand peptide plane flips that occur on a timescale of ≈100 μs and lead to global changes in the overall pleat/flatness of the CTD, thereby altering its ability to oligomerize. These findings help forge a link between chaperone dynamics, oligomerization and anti-aggregation activity which may possibly lead to new therapeutic avenues tuned to target specific substrates., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.)

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein-substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca 2+ to yield two extended "encounter" complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 μs. State A*, however, only reaches the final complex via a collapsed intermediate B ( τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow ( τ ∼ 7 to 18 ms) conformational rearrangement to state C., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

A survey, primarily based on work in the authors' laboratory during the last 10 years, is provided of recent developments in NMR studies of exchange processes involving protein-ligand and protein-protein interactions. We start with a brief overview of the theoretical background of Dark state Exchange Saturation Transfer (DEST) and lifetime line-broadening (ΔR 2 ) NMR methodology. Some limitations of the DEST/ΔR 2 methodology in applications to molecular systems with intermediate molecular weights are discussed, along with the means of overcoming these limitations with the help of closely related exchange NMR techniques, such as the measurements of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, exchange-induced chemical shifts or rapidly-relaxing components of relaxation decays. Some theoretical underpinnings of the quantitative description of global dynamics of proteins on the surface of very high molecular weight particles (nanoparticles) are discussed. Subsequently, several applications of DEST/ΔR 2 methodology are described from a methodological perspective with an emphasis on providing examples of how kinetic and relaxation parameters for exchanging systems can be reliably extracted from NMR data for each particular model of exchange. Among exchanging systems that are not associated with high molecular weight species, we describe several exchange NMR-based studies that focus on kinetic modelling of transient pre-nucleation oligomerization of huntingtin peptides that precedes aggregation and fibril formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Human mitochondrial Hsp60 (mtHsp60) is a class I chaperonin, 51% identical in sequence to the prototypical E. coli chaperonin GroEL. mtHsp60 maintains the proteome within the mitochondrion and is associated with various neurodegenerative diseases and cancers. The oligomeric assembly of mtHsp60 into heptameric ring structures that enclose a folding chamber only occurs upon addition of ATP and is significantly more labile than that of GroEL, where the only oligomeric species is a tetradecamer. The lability of the mtHsp60 heptamer provides an opportunity to detect and visualize lower-order oligomeric states that may represent intermediates along the assembly/disassembly pathway. Using cryo-electron microscopy we show that, in addition to the fully-formed heptamer and an "inverted" tetradecamer in which the two heptamers associate via their apical domains, thereby blocking protein substrate access, well-defined lower-order oligomeric species, populated at less than 6% of the total particles, are observed. Specifically, we observe open trimers, tetramers, pentamers and hexamers (comprising ∼4% of the total particles) with rigid body rotations from one subunit to the next within ∼1.5-3.5° of that for the heptamer, indicating that these may lie directly on the assembly/disassembly pathway. We also observe a closed-ring hexamer (∼2% of the particles) which may represent an off-pathway species in the assembly/disassembly process in so far that conversion to the mature heptamer would require the closed-ring hexamer to open to accept an additional subunit. Lastly, we observe several classes of tetramers where additional subunits characterized by fuzzy electron density are caught in the act of oligomer extension., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τ c The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state., Competing Interests: The authors declare no competing interest.

Huntingtin polypeptides (htt ex1 ), encoded by exon 1 of the htt gene and containing an expanded polyglutamine tract, form fibrils that accumulate within neuronal inclusion bodies, resulting in the fatal neurodegenerative condition known as Huntington's disease. Htt ex1 comprises three regions: a 16-residue N-terminal amphiphilic domain (NT), a polyglutamine tract of variable length (Q n ), and a polyproline-rich domain containing two polyproline tracts. The NT region of htt ex1 undergoes prenucleation transient oligomerization on the sub-millisecond time scale, resulting in a productive tetramer that promotes self-association and nucleation of the polyglutamine tracts. Here we show that binding of Fyn SH3, a small intracellular proline-binding domain, to the first polyproline tract of htt ex1 Q 35 inhibits fibril formation by both NMR and a thioflavin T fluorescence assay. The interaction of Fyn SH3 with htt ex1 Q 7 was investigated using NMR experiments designed to probe kinetics and equilibria at atomic resolution, including relaxation dispersion, and concentration-dependent exchange-induced chemical shifts and transverse relaxation in the rotating frame. Sub-millisecond exchange between four species is demonstrated: two major states comprising free (P) and SH3-bound (PL) monomeric htt ex1 Q 7 , and two sparsely populated dimers in which either both subunits (P 2 L 2 ) or only a single subunit (P 2 L) is bound to SH3. Binding of SH3 increases the helical propensity of the NT domain, resulting in a 25-fold stabilization of the P 2 L 2 dimer relative to the unliganded P 2 dimer. The P 2 L 2 dimer, in contrast to P 2 , does not undergo any detectable oligomerization to a tetramer, thereby explaining the allosteric inhibition of htt ex1 fibril formation by Fyn SH3.

Huntington's disease arises from polyQ expansion within the exon-1 region of huntingtin (htt ex1 ), resulting in an aggregation-prone protein that accumulates in neuronal inclusion bodies. We investigate the interaction of various htt ex1 constructs with the bacterial analog (GroEL) of the human chaperonin Hsp60. Using fluorescence spectroscopy and electron and atomic force microscopy, we show that GroEL inhibits fibril formation. The binding kinetics of htt ex1 constructs with intact GroEL and a mini-chaperone comprising the apical domain is characterized by relaxation-based NMR measurements. The lifetimes of the complexes range from 100 to 400 μs with equilibrium dissociation constants (K D ) of ∼1-2 mM. The binding interface is formed by the N-terminal amphiphilic region of htt ex1 (which adopts a partially helical conformation) and the H and I helices of the GroEL apical domain. Sequestration of monomeric htt ex1 by GroEL likely increases the critical concentration required for fibrillization., (© 2021 Wiley-VCH GmbH.)

The dynamics of methyl-bearing side chains in proteins were probed by 13 C relaxation measurements of a number of 13 C magnetization modes in selectively 13 CH 3 -labeled methyl groups of proteins. We first show how 13 C magnetization modes in a 13 CH 3 spin-system can be isolated using acute-angle 1 H radio-frequency pulses. The parameters of methyl-axis dynamics, a measure of methyl-axis ordering ( S axis 2 ) and the correlation time of fast local methyl-axis motions (τ f ), derived from 13 C relaxation in 13 CH 3 groups are compared with their counterparts obtained from 13 C relaxation in 13 CHD 2 methyl isotopomers. We show that in high-molecular-weight proteins, excellent correlations are obtained between the [ 13 CHD 2 ]-derived S axis 2 values and those extracted from relaxation of the 13 C magnetization of the I = 1/2 manifold in 13 CH 3 methyls. In smaller proteins, a certain degree of anticorrelation is observed between the S axis 2 and τ f values obtained from 13 C relaxation of the I = 1/2 manifold magnetization in 13 CH 3 methyls. These parameters can be partially decorrelated by inclusion in the analysis of relaxation data of the I = 3/2 manifold 13 C magnetization.

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1 H/ 13 C methyl protonated proteins from 100 ml cultures in M9 ++ /D 2 O medium induced at high (OD 600  ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2 H/ 15 N/ 13 C and 1 H/ 13 C labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D 2 O medium, while using three-fold less α-ketoisovaleric (1,2,3,4- 13 C 4 ; 3,4',4',4'-d 4 ) and α-ketobutyric ( 13 C 4 ; 3,3-d 2 ) acid precursors.

Optimized selection of the slow-relaxing components of single-quantum 13 C magnetization in 13 CH 3 methyl groups of proteins using acute (< 90°) angle 1 H radio-frequency pulses, is described. The optimal selection scheme is more relaxation-tolerant and provides sensitivity gains in comparison to the experiment where the undesired (fast-relaxing) components of 13 C magnetization are simply 'filtered-out' and only 90° 1 H pulses are employed for magnetization transfer to and from 13 C nuclei. When applied to methyl 13 C single-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments for studies of chemical exchange, the selection of the slow-relaxing 13 C transitions results in a significant decrease in intrinsic (exchange-free) transverse spin relaxation rates of all exchanging species. For exchanging systems involving high-molecular-weight species, the lower transverse relaxation rates translate into an increase in the information content of the resulting relaxation dispersion profiles.

Chaperone oligomerization is often a key aspect of their function. Irrespective of whether chaperone oligomers act as reservoirs for active monomers or exhibit a chaperoning function themselves, understanding the mechanism of oligomerization will further our understanding of how chaperones maintain the proteome. Here, we focus on the class-II Hsp40, human DNAJB6b, a highly efficient inhibitor of protein self-assembly in vivo and in vitro that forms functional oligomers. Using single-quantum methyl-based relaxation dispersion NMR methods we identify critical residues for DNAJB6b oligomerization in its C-terminal domain (CTD). Detailed solution NMR studies on the structure of the CTD showed that a serine/threonine-rich stretch causes a backbone twist in the N-terminal β strand, stabilizing the monomeric form. Quantitative analysis of an array of NMR relaxation-based experiments (including Carr-Purcell-Meiboom-Gill relaxation dispersion, off-resonance R 1ρ profiles, lifetime line broadening, and exchange-induced shifts) on the CTD of both wild type and a point mutant (T142A) within the S/T region of the first β strand delineates the kinetics of the interconversion between the major twisted-monomeric conformation and a more regular β strand configuration in an excited-state dimer, as well as exchange of both monomer and dimer species with high-molecular-weight oligomers. These data provide insights into the molecular origins of DNAJB6b oligomerization. Further, the results reported here have implications for the design of β sheet proteins with tunable self-assembling properties and pave the way to an atomic-level understanding of amyloid inhibition., Competing Interests: The authors declare no competing interest.