Theodoros K. Karamanos

Hydrogen–deuterium exchange (HDX) of protein backbone amides provides a powerful probe of conformational dynamics. However, when experiments are performed in H₂O/D₂O mixtures, quantitative interpretation is hindered by back exchange and isotope effects not captured by the classical Linderstro̷m–Lang (LL) model. We introduce a generalized Linderstro̷m–Lang (GLL) framework that explicitly accounts for forward and reverse exchange and for changes in protection upon isotopic substitution. Analytical solutions describe equilibrium enrichment (fractionation) and protection factors in mixtures, reducing to the LL model in pure D₂O. Application to HDX/NMR of the molecular chaperone DNAJB1 in 50% D₂O demonstrates that the GLL model recovers protection factors at 100% D₂O. Ignoring back exchange (i.e., using the LL model), protection factors are systematically underestimated. A particularly powerful feature of our approach is that a single HDX experiment in a mixture (e.g., 50% D₂O) simultaneously provides protection factors that report on conformational dynamics and local stability and fractionation factors that are sensitive to the local hydrogen-bonding environment.

By expanding fully coarse-grained MD simulations to capture local secondary structure propensities as determined by NMR chemical shifts, accurate interdomain contact maps are generated between a disordered region and a folded domain.

Intrinsically disordered, low-complexity regions frequently cooperate with folded domains to mediate protein–protein interactions, yet accurately describing these mixed folded–disordered systems remains challenging. To visualize these mixed folded proteins, experimentally guided coarse-grained (CG) molecular dynamics simulations are often employed to extend the timescales required to capture the complex dynamics in play. However, the minimalistic nature of these approaches often compromises structural accuracy and can lead to inaccurate inter-domain interactions. Here we introduce backbone dihedral terms directly derived from NMR chemical shift data in CG-simulations to characterize the open state of a mixed-folded construct of the anti-aggregation chaperone DNAJB6 that contains a folded J-domain and a disordered GF linker. By tuning residue-specific backbone dihedral parameters to match NMR-derived secondary-structure propensities of the linker in CG-simulations, we generate conformational ensembles that yield accurate interdomain contact maps. In agreement with analysis of NMR relaxation data, the resulting ensembles show that even in the nominally open state the linker experiences motions that resemble those of the closed state driven by hydrophobic residues in GF. More generally we show that by expanding CG-simulations to allow them to capture both local and global structural properties, physically relevant interdomain contacts can be retrieved.

J-domain proteins ( JDPs) act as major regulators of the proteostasis network by driving the specificity of the Hsp70 machine. Their important functions are mediated by a low-complexity glycine-/phenylalanine-rich region (GF-linker) that links the folded J-domain with the substrate binding domain. Recently, we and others have shown that in an autoinhibited JDP state, an α-helix formed within the GF-linker blocks the Hsp70 binding site on the J-domain. However, the role of the disordered GF-linker in autoinhibition and how the latter is released, are still not understood. Here, using autoinhibited DNAJB1 and DNAJB6 constructs, we show that in combination with the J-domain, the GF-linker creates a hydrophobic, partially collapsed cluster that shows a remarkable degree of long-range structural communication, disruption of which can lead to destabilisation of autoinhibition. Apart from this crucial intramolecular role, we reveal that the GF-linker can also be recognised by the substrate-binding domain of Hsp70 and dictate the lifetime of the entire JDP–Hsp70 complex. Strikingly, the GF-linkers of DNAJB1 and DNAJB6 display distinct structural properties that lead to different Hsp70 binding kinetics, showing that the behaviour of the GF-linker can vary dramatically even within the same class of JDPs.

J-domain proteins (JDPs) act as major regulators of the proteostasis network by driving the specificity of the Hsp70 machine. Their important functions are mediated by a low-complexity glycine-/phenylalanine-rich region (GF-linker) that links the folded J-domain with the substrate binding domain. Recently, we and others have shown that in an autoinhibited JDP state, an α-helix formed within GF blocks the Hsp70 binding site on the J-domain. However, the role of the disordered GF-linker in autoinhibition and how the latter is released, are still not understood. Here, using autoinhibited DNAJB1 and DNAJB6 constructs, we show that in combination with the J-domain, the GF-linker creates a hydrophobic, partially collapsed cluster that shows a remarkable degree of allosteric communication, disruption of which can lead to destabilisation of autoinhibition. Apart from this crucial intramolecular role, we reveal that the GF-linker can also be recognised by the substrate-binding domain of Hsc70 and dictate the lifetime of the entire JDP–Hsc70 complex. Importantly, both the intra- and intermolecular GF-linker functions are DNAJB-class member-specific.

Systematic backbone N-methylation of a SIM peptide results in faster, tighter binging to SUMO driven by a reduced entropy of activation; this opens new opportunities to develop inhibitors of β-strand mediated protein–protein interactions.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs and templates for PPI modulator development. Using the interaction between small ubiquitin-like modifiers (SUMO) and SUMO-interacting motifs (SIMs), here we show that N-methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition. Backbone N-methylation in appropriate locations results in faster target binding, and thus higher affinity, as shown by fluorescence anisotropy, relaxation-based NMR experiments, and computational analysis. We show that such higher affinities occur as a consequence of an increase in the energy of the unbound state, and a reduction in the entropic contribution to the binding and activation energies. Thus, backbone N-Methylation may represent a useful modification within the peptidomimetic toolbox to probe β-strand mediated coupled binding and folding interactions.

Coevolution between protein residues is normally interpreted as direct contact. However, the evolutionary record of a protein sequence contains rich information that may include long‐range functional couplings, couplings that report on homo‐oligomeric states or even conformational changes. Due to the complexity of the sequence space and the lack of structural information on various members of a protein family, it has been difficult to effectively mine the additional information encoded in a multiple sequence alignment (MSA). Here, taking advantage of the recent release of the AlphaFold (AF) database we attempt to identify coevolutionary couplings that cannot be explained simply by spatial proximity. We propose a simple computational method that performs direct coupling analysis on a MSA and searches for couplings that are not satisfied in any of the AF models of members of the identified protein family. Application of this method on 2012 protein families suggests that ~12% of the total identified coevolving residue pairs are spatially distant and more likely to be disordered than their contacting counterparts. We expect that this analysis will help improve the quality of coevolutionary distance restraints used for structure determination and will be useful in identifying potentially functional/allosteric cross‐talk between distant residues.

Degenerate spin-systems consisting of magnetically equivalent nuclear spins, such as a ¹H₃ spin-system in selectively ¹³CH₃-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 ¹H CPMG relaxation dispersion profiles obtained for (¹³C)¹H₃ groups. We describe an alternative, experimentally simple solution and design a "steady-state" methyl ¹H CPMG scheme, where 90° or acute-angle (1H radiofrequency pulses are applied after each CPMG echo in-phase with methyl ¹H 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.