Application of native mass spectrometry in studying intrinsically disordered proteins: A special focus on neurodegenerative diseases

Intrinsically disordered proteins (IDPs) are integral part of the proteome, regulating vital biological processes. Such proteins gained further visibility due to their key role in neurodegenerative diseases and cancer. IDPs however, escape structural characterization by traditional biophysical tools owing to their extreme flexibility and heterogeneity. In this review, we discuss the advantages of native mass spectrometry (MS) in analysing the atypical conformational dynamics of IDPs and recent advances made in the field. Especially, MS studies unravelling the conformational facets of IDPs involved in neurodegenerative diseases are highlighted. The limitations and the future promises of native MS while studying IDPs have been discussed.     

Thinking outside the crystal: complementary approaches for examining transporter conformational change

As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present 1?F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.     

Thinking outside the crystal: Complementary approaches for examining transporter mechanism

As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present ¹⁹F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.     

Reweighting methods for elucidation of conformation ensembles of proteins

Proteins are inherently dynamic macromolecules that exist in equilibrium among multiple conformational states, and motions of protein backbone and side chains are fundamental to biological function. The ability to characterize the conformational landscape is particularly important for intrinsically disordered proteins, multidomain proteins, and weakly bound complexes, where single-structure representations are inadequate. As the focus of structural biology shifts from relatively rigid macromolecules toward larger and more complex systems and molecular assemblies, there is a need for structural approaches that can paint a more realistic picture of such conformationally heterogeneous systems. Here, we review reweighting methods for elucidation of structural ensembles based on experimental data, with the focus on applications to multidomain proteins.     

Thermodynamics of protein denatured states

Recent work on the thermodynamics of protein denatured states is providing insight into the stability of residual structure and the conformational constraints that affect the disordered states of proteins. Current data from native state hydrogen exchange and the pH dependence of protein stability indicate that residual structure can modulate the stability of the denatured state by up to 4 kcal mol(-1). NMR structural data have emphasized the role of hydrophobic clusters in stabilizing denatured state residual structures, however recent results indicate that electrostatic interactions, both favorable and unfavorable, are also important modulators of the stability of the denatured state. Thermodynamics methods that take advantage of histidine-heme ligation chemistry have also been developed to probe the conformational constraints that act on denatured states. These methods have provided insights into the role of excluded volume, chain stiffness, and loop persistence in modulating the conformational preferences of highly disordered proteins. New insights into protein folding and novel methods to manipulate protein stability are emerging from this work.     

Beyond the random coil: stochastic conformational switching in intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) participate in critical cellular functions that exploit the flexibility and rapid conformational fluctuations of their native state. Limited information about the native state of IDPs can be gained by the averaging over many heterogeneous molecules that is unavoidable in ensemble approaches. We used single molecule fluorescence to characterize native state conformational dynamics in five synaptic proteins confirmed to be disordered by other techniques. For three of the proteins, SNAP-25, synaptobrevin and complexin, their conformational dynamics could be described with a simple semiflexible polymer model. Surprisingly, two proteins, neuroligin and the NMDAR-2B glutamate receptor, were observed to stochastically switch among distinct conformational states despite the fact that they appeared intrinsically disordered by other measures. The hop-like intramolecular diffusion found in these proteins is suggested to define a class of functionality previously unrecognized for IDPs.     

Discrete Molecular Dynamics Can Predict Helical Prestructured Motifs in Disordered Proteins

Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available.     

Errata

Abstract

Mass spectrometry (MS)-based proteomics is an unrivaled tool for studying complex biological systems and diseases in the post-genomic era. In recent years, MS has emerged as a powerful structural biological tool to characterize protein conformation and conformational dynamics. The advantages of MS in structural studies are most evident for membrane proteins such as GPCRs (G protein-coupled receptors), where other well-established structural methods such as X-ray crystallography and NMR remain challenging. For proteins with available high-resolution structures, MS-based structural strategies can provide valuable, previously inaccessible information on protein conformational changes and dynamics, protein motion/flexibility, ligand–protein binding, and protein–protein interfaces. In the past several years, we have developed and adapted a number of MS-based structural approaches, such as CDSiL-MS (Conformational changes and Dynamics using Stable-isotope Labeling and MS), CXMS (Crosslinking/MS) and HDXMS (Hydrogen-Deuterium Exchange MS), to study protein structures and conformational dynamics in human β2-adrenegic receptor (β2AR) signaling. In this mini-review, we will highlight several examples demonstrating the power of MS in structural analysis to better elucidate the structural basis of GPCR signaling, particularly through the β-arrestin-mediated GPCR signaling pathway.     

Order propensity of an intrinsically disordered protein,the cyclin‐dependent‐kinase inhibitor Sic1

Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin‐dependent‐kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray‐ionization mass spectrometry (ESI‐MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or premolten globules. Proteolysis‐hypersensitive sites cluster at the N‐terminus and in the middle of the molecule, whereas the most structured region resides at the C‐terminus, including part of the inhibitory domain and the casein‐kinase‐2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein. Proteins 2009;76:731–746. © 2009 Wiley‐Liss, Inc.     

Towards the physical basis of how intrinsic disorder mediates protein function

Intrinsically disordered proteins (IDPs) are an important class of functional proteins that is highly prevalent in biology and has broad association with human diseases. In contrast to structured proteins, free IDPs exist as heterogeneous and dynamical conformational ensembles under physiological conditions. Many concepts have been discussed on how such intrinsic disorder may provide crucial functional advantages, particularly in cellular signaling and regulation. Establishing the physical basis of these proposed phenomena requires not only detailed characterization of the disordered conformational ensembles, but also mechanistic understanding of the roles of various ensemble properties in IDP interaction and regulation. Here, we review the experimental and computational approaches that may be integrated to address many important challenges of establishing a "structural" basis of IDP function, and discuss some of the key emerging ideas on how the conformational ensembles of IDPs may mediate function, especially in coupled binding and folding interactions.     

Native ion mobility-mass spectrometry and related methods in structural biology

Mass spectrometry-based methods have become increasingly important in structural biology — in particular for large and dynamic, even heterogeneous assemblies of biomolecules. Native electrospray ionization coupled to ion mobility-mass spectrometry provides access to stoichiometry, size and architecture of noncovalent assemblies; while non-native approaches such as covalent labeling and H/D exchange can highlight dynamic details of protein structures and capture intermediate states. In this overview article we will describe these methods and highlight some recent applications for proteins and protein complexes, with particular emphasis on native MS analysis. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Utilization of protein intrinsic disorder knowledge in structural proteomics

Intrinsically disordered proteins (IDPs) and proteins with long disordered regions are highly abundant in various proteomes. Despite their lack of well-defined ordered structure, these proteins and regions are frequently involved in crucial biological processes. Although in recent years these proteins have attracted the attention of many researchers, IDPs represent a significant challenge for structural characterization since these proteins can impact many of the processes in the structure determination pipeline. Here we investigate the effects of IDPs on the structure determination process and the utility of disorder prediction in selecting and improving proteins for structural characterization. Examination of the extent of intrinsic disorder in existing crystal structures found that relatively few protein crystal structures contain extensive regions of intrinsic disorder. Although intrinsic disorder is not the only cause of crystallization failures and many structured proteins cannot be crystallized, filtering out highly disordered proteins from structure-determination target lists is still likely to be cost effective. Therefore it is desirable to avoid highly disordered proteins from structure-determination target lists and we show that disorder prediction can be applied effectively to enrich structure determination pipelines with proteins more likely to yield crystal structures. For structural investigation of specific proteins, disorder prediction can be used to improve targets for structure determination. Finally, a framework for considering intrinsic disorder in the structure determination pipeline is proposed.     

Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering

Small-angle scattering of X-rays (SAXS) is an established method to study the overall structure and structural transitions of biological macromolecules in solution. For folded proteins, the technique provides three-dimensional low resolution structures ab initio or it can be used to drive rigid-body modeling. SAXS is also a powerful tool for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs), and is highly complementary to the high resolution methods of X-ray crystallography and NMR. Here we present the basic principles of SAXS and review the main approaches to the characterization of IDPs and flexible multidomain proteins using SAXS. Together with the standard approaches based on the analysis of overall parameters, a recently developed Ensemble Optimization Method (EOM) is now available. The latter method allows for the co-existence of multiple protein conformations in solution compatible with the scattering data. Analysis of the selected ensembles provides quantitative information about flexibility and also offers insights into structural features. Examples of the use of SAXS and combined approaches with NMR, X-ray crystallography, and computational methods to characterize completely or partially disordered proteins are presented.     

Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.     

Distance-Based Metrics for Comparing Conformational Ensembles of Intrinsically Disordered Proteins

Intrinsically disordered proteins are proteins whose native functional states represent ensembles of highly diverse conformations. Such ensembles are a challenge for quantitative structure comparisons because their conformational diversity precludes optimal superimposition of the atomic coordinates necessary for deriving common similarity measures such as the root mean-square deviation of these coordinates. Here, we introduce superimposition-free metrics that are based on computing matrices of the Cα-Cα distance distributions within ensembles and comparing these matrices between ensembles. Differences between two matrices yield information on the similarity between specific regions of the polypeptide, whereas the global structural similarity is captured by the root mean-square difference between the medians of the Cα-Cα distance distributions of two ensembles. Together, our metrics enable rigorous investigations of structure-function relationships in conformational ensembles of intrinsically disordered proteins derived using experimental restraints or by molecular simulations and for proteins containing both structured and disordered regions.     

Guiding probabilistic search of the protein conformational space with structural profiles

The roughness of the protein energy surface poses a significant challenge to search algorithms that seek to obtain a structural characterization of the native state. Recent research seeks to bias search toward near-native conformations through one-dimensional structural profiles of the protein native state. Here we investigate the effectiveness of such profiles in a structure prediction setting for proteins of various sizes and folds. We pursue two directions. We first investigate the contribution of structural profiles in comparison to or in conjunction with physics-based energy functions in providing an effective energy bias. We conduct this investigation in the context of Metropolis Monte Carlo with fragment-based assembly. Second, we explore the effectiveness of structural profiles in providing projection coordinates through which to organize the conformational space. We do so in the context of a robotics-inspired search framework proposed in our lab that employs projections of the conformational space to guide search. Our findings indicate that structural profiles are most effective in obtaining physically realistic near-native conformations when employed in conjunction with physics-based energy functions. Our findings also show that these profiles are very effective when employed instead as projection coordinates to guide probabilistic search toward undersampled regions of the conformational space.