20 research outputs found
New experimental and theoretical tools for studying protein systems with elements of structural disorder
Disordered proteins are one class of proteins which do not possess well-folded three-dimensional structures as their native conformations. Many eukaryotic proteins have been found to be fully disordered or contain certain disordered regions. Disordered proteins usually display several characteristic properties, such as increased motional freedom and the conformational heterogeneity caused by that. The elements of structural disorder are commonly involved in many important biological functions and are implicated in many diseases. Therefore, the study of disordered proteins has become one of the most important research topics in recent years. This thesis presents results from three different research projects; the common feature is that all systems being studied contain varying amount of structural disorder. Most results have been obtained based on experimental nuclear magnetic resonance (NMR) studies and molecular dynamics (MD) simulations. Both are among the most popular biophysical techniques for studying molecular dynamics. The first project investigates the relationship between domain cooperativity and residual dipolar coupling (RDC) parameters based on a series of two-domain chimera proteins with disordered linkers. Many eukaryotic proteins contain multiple domains and their biological functions are closely related to the property of domain cooperativity, which is often regulated by the linker region. Therefore it is necessary to develop suitable tools to characterize linker region properties in order to better understand biological functions of multidomain proteins. The second project is about the development of NMR pulse sequences for studying disordered proteins. Two new NMR pulse sequences, PD-CPMG and CP-HISQC, have been developed. Both experiments are well suited for studying intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) under physiological conditions. These two experiments produce higher precision for 15N R2 rates measurement or higher sensitivity in 1H– 15N HSQC spectra respectively. Besides, they also show many advantages over most other existing experiments for studying IDPs. The last project is about protein-peptide encounter complex study based on Crk-Sos model system. The ten-residue Sos peptide serves as a minimal model for disordered proteins. Encounter complex is an important type of intermediate state formed during many protein interactions. Such complexes are usually characterized by a large amount of motional freedom and conformational heterogeneity. Therefore their properties are considerably different from tight-binding complexes which are more commonly studied. Although it is usually quite difficult to study encounter complexes using standard biophysical techniques, in this project we have successfully characterized structural and dynamic properties of Crk-Sos electrostatic encounter complex with a combination of MD simulations and experimental NMR approaches. It can be directly seen from the structural model based on MD trajectories that Sos peptide in the encounter complex remains highly dynamic, sampling large area on the surface of Crk N-SH3 domain. Such strategy can also be utilized for studying many other encounter complexes involving disordered proteins or peptide
The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.
Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes
Exploring methods to expedite the recording of CEST datasets using selective pulse excitation
International audienc
Dramatic Decrease in CEST Measurement Times Using Multi-Site Excitation
International audienc
Towards autonomous analysis of Chemical Exchange Saturation Transfer experiments using Deep Neural Networks
Macromolecules often exchange between functional states on timescales that can be accessed with NMR spectroscopy and many NMR tools have been developed to characterise the kinetics and thermodynamics of the exchange processes, as well as the structure of the conformers that are involved. However, analysis of the NMR data that report on exchanging macromolecules often hinges on complex least-squares fitting procedures as well as human experience and intuition, which, in some cases, limits the widespread use of the methods. The applications of deep neural networks (DNNs) and artificial intelligence have increased significantly in the sciences, and recently, specifically, within the field of biomolecular NMR, where DNNs are now available for tasks such as the reconstruction of sparsely sampled spectra, peak picking, and virtual decoupling. Here we present a DNN for the analysis of chemical exchange saturation transfer (CEST) data reporting on two- or three-site chemical exchange involving sparse state lifetimes of between approximately 3 - 60 ms, the range most frequently observed via experiment. The work presented here focuses on the 1H CEST class of methods that are further complicated, in relation to applications to other nuclei, by anti-phase features. The developed DNNs accurately predict the chemical shifts of nuclei in the exchanging species directly from anti-phase 1HN CEST profiles, along with an uncertainty associated with the predictions. The performance of the DNN was quantitatively assessed using both synthetic and experimental anti-phase CEST profiles. The assessments show that the DNN accurately determines chemical shifts and their associated uncertainties. The DNNs developed here do not contain any parameters for the end-user to adjust and the method therefore allows for autonomous analysis of complex NMR data that report on conformational exchange
Role of Electrostatic Interactions in Binding of Peptides and Intrinsically Disordered Proteins to Their Folded Targets: 2. The Model of Encounter Complex Involving the Double Mutant of the c‑Crk N‑SH3 Domain and Peptide Sos
In
the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014, 53, 6473), we have studied the
complex between the 10-residue peptide Sos
and N-terminal SH3 domain from adaptor protein c-Crk. In the second
part (this paper), we designed the double mutant of the c-Crk N-SH3
domain, W169F/Y186L, with the intention to eliminate the interactions
responsible for tight peptide–protein binding, while retaining
the interactions that create the initial electrostatic encounter complex.
The resulting system was characterized experimentally by measuring
the backbone and side-chain <sup>15</sup>N relaxation rates, as well
as binding shifts and <sup>1</sup>H<sup>N</sup> temperature coefficients.
In addition, it was also modeled via a series of ∼5 μs
molecular dynamics (MD) simulations recorded in a large water box
under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have
found that the strength of arginine-aspartate and arginine-glutamate
salt bridges is overestimated in the original force field. To address
this problem we have applied the empirical force-field correction
described in paper 1. Specifically, the Lennard-Jones equilibrium
distance for the nitrogen–oxygen pair across Arg-to-Asp/Glu
salt bridges has been increased by 3%. This modification led to MD
models in good agreement with the experimental data. The emerging
picture is that of a fuzzy complex, where the peptide “dances”
over the surface of the protein, making transient contacts via salt-bridge
interactions. Every once in a while the peptide assumes a certain
more stable binding pose, assisted by a number of adventitious polar
and nonpolar contacts. On the other hand, occasionally Sos flies off
the protein surface; it is then guided by electrostatic steering to
quickly reconnect with the protein. The dynamic interaction between
Sos and the double mutant of c-Crk N-SH3 gives rise to only small
binding shifts. The peptide retains a high degree of conformational
mobility, although it is appreciably slowed down due to its (loose)
association with the protein. Note that spin relaxation data are indispensable
in determining the dynamic status of the peptide. Such data can be
properly modeled only on a basis of <i>bona fide</i> MD
simulations, as shown in our study. We anticipate that in future the
field will move away from the ensemble view of protein disorder and
toward more sophisticated MD models. This will require further optimization
of force fields, aimed specifically at disordered systems. Efforts
in this direction have been recently initiated by several research
groups; the empirical salt-bridge correction proposed in our work
falls in the same category. MD models obtained with the help of suitably
refined force fields and guided by experimental NMR data will provide
a powerful insight into an intricate world of disordered biomolecules
Measuring Solvent Hydrogen Exchange Rates by Multifrequency Excitation 15 N CEST: Application to Protein Phase Separation
International audienc
Probing Conformational Exchange in Weakly Interacting, Slowly Exchanging Protein Systems via Off-Resonance <i>R</i><sub>1ρ</sub> Experiments: Application to Studies of Protein Phase Separation
<i>R</i><sub>1ρ</sub> relaxation dispersion experiments
are increasingly used in studies of protein dynamics on the micro-
to millisecond time scale. Traditional <i>R</i><sub>1ρ</sub> relaxation dispersion approaches are typically predicated on changes
in chemical shifts between corresponding probe spins, <i>Δω</i><sub>GE</sub>, in the interconverting states. Here, we present a
new application of off-resonance <sup>15</sup>N <i>R</i><sub>1ρ</sub> relaxation dispersion that enables the quantification
of slow exchange processes even in the limit where <i>Δω</i><sub>GE</sub> = 0 so long as the spins in the exchanging states have
different intrinsic transverse relaxation rates (<i>ΔR</i><sub>2</sub> = <i>R</i><sub>2,E</sub> – <i>R</i><sub>2,G</sub> ≠ 0). In this limit, the dispersion
profiles become inverted relative to those measured in the case where <i>Δω</i><sub>GE</sub> ≠ 0, <i>ΔR</i><sub>2</sub> = 0. The theoretical background to understand this effect
is presented, along with a simplified exchange matrix that is valid
in the limits that are germane here. An application to the study of
the dynamics of the germ granule protein Ddx4 in a highly concentrated
phase-separated state is described. Notably, exchange-based dispersion
profiles can be obtained despite the fact that <i>Δω</i><sub>GE</sub> ≈ 0 and <i>ΔR</i><sub>2</sub> is small, ∼20–30 s<sup>–1</sup>. Our results
are consistent with the formation of a significantly populated excited
conformational state that displays increased contacts between adjacent
protein molecules relative to the major conformer in solution, leading
to a decrease in overall motion of the protein backbone. A complete
set of exchange parameters is obtained from analysis of a single set
of <sup>15</sup>N off-resonance <i>R</i><sub>1ρ</sub> measurements recorded at a single static magnetic field and with
a single spin-lock radio frequency field strength. This new approach
holds promise for studies of weakly interacting systems, especially
those involving intrinsically disordered proteins that form phase-separated
organelles, where little change to chemical shifts between interconverting
states would be expected, but where finite <i>ΔR</i><sub>2</sub> values are observed
Measuring Diffusion Constants of Invisible Protein Conformers by Triple-Quantum H-1 CPMG Relaxation Dispersion
Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin-relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG-based experiment for measuring translational diffusion constants of invisible states using a pulsed-field gradient approach that exploits methyl H-1 triple-quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state
Opening of a cryptic pocket in β-lactamase increases penicillinase activity
Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket\u27s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets