SignificanceDevelopment of specialized instrumentation enables rapid switching of the hydrostatic pressure inside an operating NMR spectrometer. This technology allows observation of protein signals during the repeated folding process. Applied to ubiquitin, a previously extensively studied model of protein folding, the methodology reveals an initially highly dynamic state that deviates relatively little from random coil behavior but also provides evidence for numerous repeatedly failed folding events, previously only observed in computer simulations. Above room temperature, direct NMR evidence shows a ∼50% fraction of proteins folding through an on-pathway kinetic intermediate, thereby revealing two equally efficient parallel folding pathways.

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy.

Protein misfolding and aggregation are pathological events that place a significant amount of stress on the maintenance of protein homeostasis (proteostasis). To prevent and repair protein misfolding and aggregation, cells are equipped with robust mechanisms that mainly rely on molecular chaperones. Two classes of molecular chaperones, heat shock protein 70 kDa (Hsp70) and Hsp40, recognize and bind to misfolded proteins, preventing their toxic biomolecular aggregation and enabling refolding or targeted degradation. Here, we review the current state of structural biology of Hsp70 and Hsp40-Hsp70 complexes and examine the link between their structures, dynamics, and functions. We highlight the power of nuclear magnetic resonance (NMR) spectroscopy to untangle complex relationships behind molecular chaperones and their mechanism(s) of action.

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Deep learning-based approaches to protein structure prediction, such as AlphaFold2 and RoseTTAFold, can now define many protein structures with atomic-level accuracy. The AlphaFold Protein Structure Database (AFDB) contains a predicted structure for nearly every protein in the human proteome, including proteins that have intrinsically disordered regions (IDRs), which do not adopt a stable structure and rapidly interconvert between conformations. Although it is generally assumed that IDRs have very low AlphaFold2 confidence scores that reflect low-confidence structural predictions, we show here that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. The amino-acid sequences of IDRs with high-confidence structures do not show significant similarity to the Protein Data Bank; instead, these IDR sequences exhibit a higher degree of positional amino-acid sequence conservation and are more enriched in charged and hydrophobic residues than IDRs with low-confidence structures. We compared the AlphaFold2 predictions to experimental NMR data for a subset of IDRs known to fold under specific conditions, finding that AlphaFold2 tends to capture the folded state structure. We note, however, that these AlphaFold2 predictions cannot detect functionally relevant structural plasticity within IDRs and cannot offer an ensemble representation of IDRs. Nevertheless, AlphaFold2 assigns high-confidence scores to about 60% of a set of 350 IDRs that have been reported to conditionally fold, suggesting that AlphaFold2 has learned to identify conditionally folded IDRs, which is unexpected, since IDRs were minimally represented in the training data. Leveraging this ability to discover IDRs that conditionally fold, we find that up to 80% of IDRs in archaea and bacteria are predicted to conditionally fold, but less than 20% of eukaryotic IDRs. Our results suggest that a large majority of IDRs in the proteomes of human and other eukaryotes would be expected to function in the absence of conditional folding.

In unfolded proteins, peptideb onds involving Pro residues exist in equilibrium between the minor cis and major trans conformations. Foldedp roteins predominantly contain transPro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be ar ate-limiting step in protein folding. Moreover,k inases and phosphatases that act upon Ser/ThrÀPro motifs exhibit preferential recognition of either the cis-o rtrans-Pro conformer.H ere, NMR spectrao btained at both atmospherica nd high pressures indicate that the population of cis-Pro falls wellb elow previous estimates, an effect attributed to the use of short peptides with charged terminii n most prior model studies. For the intrinsically disordered protein a-synuclein, cis-Pro populations at all of its five XÀPro bonds are less than 5%,w ith only modest ionic strength dependence and no detectable effecto ft he previously demonstrated interaction between the N-and C-terminal halveso f the protein.Comparison to small peptides with the same amino-acid sequence indicates that peptides, particularly those with unblocked, oppositely charged amino and carboxyl end groups,s trongly overestimate the amount of cis-Pro.Within proteins, the vast majority (> 99.5 %) of peptideb onds not involvingp rolinee xist in the trans conformation,i nw hich the dihedral angle (w)i s1 808.T he lowly populated cis conformer requires 1808 rotation about the planar CO(iÀ1)ÀN(i)p eptide bond (w = 08), but such ar otation induces steric clash between the C a (iÀ1) and C a (i)a toms.T hisc reatesafree-energy difference between the trans and cis conformationso fa pproximately 2-6 kcal mol À1 in non-Pro peptideb onds, and ah igh energy barriertor otation of the partial double bond ( % 20 kcal mol À1 )o verwhelmingly favors the trans state. [1][2][3] However,i n peptideb onds between any amino acid (X) and proline( X À Pro), the trans and cis conformers have as ubstantially lower energy difference owing to the cyclic nature of the proline side chain. Thus, cis-peptidyl-prolyl (cis-Pro) conformationsi n unfolded polypeptide chains are populated to significantly higher levels, with values that range from 5t o8 0% in model peptides, [4][5][6][7][8][9][10][11][12][13] depending on the precise amino-acid composition, with virtually no detectable dependence on temperature. [11,14] In folded proteins, local interactions aroundX ÀPro bonds typically induce1 00 %p opulation of either the cis or trans conformation. [15][16][17] cis-Pro bonds and their slow isomerization to the trans state, approximately1 0 À3 to 10 À2 s À1 at room temperature, depending on the types of adjacent residues, [12] can be the rate-limiting step in protein folding, [2] as most non-native cis-Pro bonds in the unfolded protein require isomerization to the native trans conformations for folding to proceed. Indeed, ac lass of molecular chaperones has evolved to catalyze cis-trans proline isomerization in nascent polypeptides, [18] and in vitro refolding studies have demonstrated that such peptidyl-prolyl isomerases...

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved -crystallin domain, and provide the first structural characterization of a small heat-shock protein monomer. While HSP27 assembles into oligomers, we show that the transiently populated monomers released upon reduction are highly active chaperones in vitro, but are kinetically unstable and susceptible to uncontrolled aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance spectroscopy, we reveal that the pair of strands that mediate dimerization become partially disordered in the monomer. Strikingly, we note that numerous HSP27 mutations associated with inherited neuropathies cluster to this unstructured region. The high degree of sequence conservation in the -crystallin domain amongst mammalian sHSPs suggests that partially unfolded monomers may be a general, functional feature of these molecular chaperones.

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.