Structural context modulates the conformational ensemble of the intrinsically disordered amino terminus of α-synuclein.

Regions of intrinsic disorder play crucial roles in biological systems, yet they often elude characterization by conventional biophysical techniques. To capture conformational distributions across different timescales, we employed a freezing approach coupled with solid-state NMR analysis. Using segmentally isotopically labeled α-synuclein (α-syn), we investigated the conformational preferences of the six alanines, three glycines, and a single site (L8) in the disordered amino terminus under three distinct conditions: in 8 M urea, as a frozen monomer in buffer, and within the disordered regions flanking the amyloid core.

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited.

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells.

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble.

Conformational ensembles explain NMR spectra of frozen intrinsically disordered proteins.

Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble.

Visualizing protein breathing motions associated with aromatic ring flipping.

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events.

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown.

Enthalpy-Entropy Compensation in the Promiscuous Interaction of an Intrinsically Disordered Protein with Homologous Protein Partners.

Intrinsically disordered proteins (IDPs) can engage in promiscuous interactions with their protein targets; however, it is not clear how this feature is encoded in the primary sequence of the IDPs and to what extent the surface properties and the shape of the binding cavity dictate the binding mode and the final bound conformation. Here we show, using a combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), that the promiscuous interaction of the intrinsically disordered regulatory domain of the mitogen-activated protein kinase kinase MKK4 with p38α and JNK1 is facilitated by folding-upon-binding into two different conformations, despite the high sequence conservation and structural homology between p38α and JNK1. Our results support a model whereby the specific surface properties of JNK1 and p38α dictate the bound conformation of MKK4 and that enthalpy-entropy compensation plays a major role in maintaining comparable binding affinities for MKK4 towards the two kinases.

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state.

Ensemble Calculation for Intrinsically Disordered Proteins Using NMR Parameters.

Intrinsically disordered proteins (IDPs) perform their function despite their lack of well-defined tertiary structure. Residual structure has been observed in IDPs, commonly described as transient/dynamic or expressed in terms of fractional populations. In order to understand how the protein primary sequence dictates the dynamic and structural properties of IDPs and in general to understand how IDPs function, atomic-level descriptions are needed.

Structure and dynamics of the MKK7-JNK signaling complex.

Signaling specificity in the mitogen-activated protein kinase (MAPK) pathways is controlled by disordered domains of the MAPK kinases (MKKs) that specifically bind to their cognate MAPKs via linear docking motifs. MKK7 activates the c-Jun N-terminal kinase (JNK) pathway and is the only MKK containing three motifs within its regulatory domain. Here, we characterize the conformational behavior and interaction mechanism of the MKK7 regulatory domain.

Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR.

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX.

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, has a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We define a new mechanism of allosteric inhibition that targets the C-terminal, noncatalytic segment of PTP1B.

Small-angle X-ray scattering- and nuclear magnetic resonance-derived conformational ensemble of the highly flexible antitoxin PaaA2.

Antitoxins from prokaryotic type II toxin-antitoxin modules are characterized by a high degree of intrinsic disorder. The description of such highly flexible proteins is challenging because they cannot be represented by a single structure. Here, we present a combination of SAXS and NMR data to describe the conformational ensemble of the PaaA2 antitoxin from the human pathogen E.

Conformational propensities of intrinsically disordered proteins from NMR chemical shifts.

The realization that a protein can be fully functional even in the absence of a stable three-dimensional structure has motivated a large number of studies describing the conformational behaviour of these proteins at atomic resolution. Here, we review recent advances in the determination of local structural propensities of intrinsically disordered proteins (IDPs) from experimental NMR chemical shifts. A mapping of the local structure in IDPs is of paramount importance in order to understand the molecular details of complex formation, in particular, for IDPs that fold upon binding or undergo structural transitions to pathological forms of the same protein.