A wide variety of biological processes rely upon interactions between proteins and lipids, ranging from molecular transport to the organization of the cell membrane. It was recently established that ...electrospray ionization mass spectrometry (ESI-MS) is capable of capturing transient interactions between membrane proteins and their lipid environment, and a detailed understanding of the underlying processes is therefore of high importance. Here, we apply ESI-MS to investigate the factors that govern complex formation in solution and gas phases by comparing nonselective lipid binding with soluble and membrane proteins. We find that exogenously added lipids did not bind to soluble proteins, suggesting that lipids have a low propensity to form electrospray ionization adducts. The presence of detergents at increasing micelle concentrations, on the other hand, resulted in moderate lipid binding to soluble proteins. A direct ESI-MS comparison of lipid binding to the soluble protein serum albumin and to the integral membrane protein NapA shows that soluble proteins acquire fewer lipid adducts. Our results suggest that protein–lipid complexes form via contacts between proteins and mixed lipid/detergent micelles. For soluble proteins, these complexes arise from nonspecific contacts between the protein and detergent/lipid micelles in the electrospray droplet. For membrane proteins, lipids are incorporated into the surrounding micelle in solution, and complex formation occurs independently of the ESI process. We conclude that the lipids in the resulting complexes interact predominantly with sites located in the transmembrane segments, resulting in nativelike complexes that can be interrogated by MS.
Background: Adaptive mutations that alter protein functionality are enriched within intrinsically disordered protein regions (IDRs), thus conformational flexibility correlates with evolvability. ...Pre-structured motifs (PreSMos) with transient propensity for secondary structure conformation are believed to be important for IDR function. The glucocorticoid receptor taulcore transcriptional activation domain (GR taulcore) domain contains three a-helical PreSMos in physiological buffer conditions.
Methods: Sixty change-of-function mutants affecting the intrinsically disordered 58-residue GR taulcore were studied using disorder prediction and molecular dynamics simulations.
Results: Change-of-function mutations were partitioned into seven clusters based on their effect on IDR predictions and gene activation activity. Some mutations selected from clusters characterized by mutations altering the IDR prediction score, altered the apparent stability of the a-helical form of one of the PreSMos in molecular dynamics simulations, suggesting PreSMo stabilization or destabilization as strategies for functional adaptation. Indeed all tested gain-of-function mutations affecting this PreSMo were associated with increased stability of the a-helical PreSMo conformation, suggesting that PreSMo stabilization may be the main mechanism by which adaptive mutations can increase the activity of this IDR type. Some mutations did not appear to affect PreSMo stability.
Conclusions: Changes in PreSMo stability account for the effects of a subset of change-of-function mutants affecting the GR taulcore IDR. General significance: Long IDRs occur in about 50% of human proteins. They are poorly characterized despite much recent attention. Our results suggest the importance of a subtle balance between PreSMo stability and IDR activity, which may provide a novel target for future pharmaceutical intervention.
α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. ...Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass ...spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
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•Mass spectrometry captures intact complexes of the peripheral membrane protein DHODH•Detergent removal in the gas phase reveals lipid and co-factor binding•DHODH attaches to the membrane by binding charged phospholipids•Lipids stabilize the flexible substrate- and drug-binding site
The combination of mass spectrometry and molecular dynamics simulations provides insights into the relationship between lipid and substrate binding to the peripheral membrane protein dehydroorotate dehydrogenase, revealing ligand-induced stabilization of the flexible membrane-binding region.
α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinsonâs disease patients. ...Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of â2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
Adaptive mutations that alter protein functionality are enriched within intrinsically disordered protein regions (IDRs), thus conformational flexibility correlates with evolvability. Pre-structured ...motifs (PreSMos) with transient propensity for secondary structure conformation are believed to be important for IDR function. The glucocorticoid receptor tau1core transcriptional activation domain (GR tau1core) domain contains three α-helical PreSMos in physiological buffer conditions.
Sixty change-of-function mutants affecting the intrinsically disordered 58-residue GR tau1core were studied using disorder prediction and molecular dynamics simulations.
Change-of-function mutations were partitioned into seven clusters based on their effect on IDR predictions and gene activation activity. Some mutations selected from clusters characterized by mutations altering the IDR prediction score, altered the apparent stability of the α-helical form of one of the PreSMos in molecular dynamics simulations, suggesting PreSMo stabilization or destabilization as strategies for functional adaptation. Indeed all tested gain-of-function mutations affecting this PreSMo were associated with increased stability of the α-helical PreSMo conformation, suggesting that PreSMo stabilization may be the main mechanism by which adaptive mutations can increase the activity of this IDR type. Some mutations did not appear to affect PreSMo stability.
Changes in PreSMo stability account for the effects of a subset of change-of-function mutants affecting the GR tau1core IDR.
Long IDRs occur in about 50% of human proteins. They are poorly characterized despite much recent attention. Our results suggest the importance of a subtle balance between PreSMo stability and IDR activity, which may provide a novel target for future pharmaceutical intervention.
•The disordered GRtau1core activation domain contains pre-structured α-helices.•Effects on activity and predicted disorder allow clustering of a set of 60 mutants.•Several clusters are characterized by mutants that affect pre-structured region helicity.•α-Helicity changes of pre-structured regions are an important strategy for adaptation.•Other strategies must account for adaptation in other mutant clusters.
In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to ...inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.
Intrinsically Disordered Proteins have regions that lack a stable structure under physiological conditions, yet they constitute an ensemble of conformations. Intrinsically Disordered Proteins are ...present in many biological functions, like the regulation of transcription. The altered regulation of Intrinsically Disordered Proteins is associated with many human diseases. The possibility of inhibiting Intrinsically Disordered Proteins is of great importance to chemical biology and drug discovery. Transcription Factors, like Glucocorticoid Receptor and c-Myc, are examples of proteins that contain large disordered regions. c-Myc disordered regions are associated with Burkitt’s lymphoma and therefore it is considered a promising drug target.In this work we studied, by Molecular Dynamics simulations, a peptide fragment (from residue 187 to 202) of a disordered region of Glucocorticoid Receptor. Using the same methodology, we chose a peptide fragment (from residue 42 to 63) of a disordered region of c-Myc, to study it alone and with a ligand, which represents a model drug compound against Lymphoma. The major findings from this work are:• The data on the fragment of the Glucocorticoid Receptor showed an inverse correlation between relative activity and disorder;• Single mutation of peptide fragments of the Glucocorticoid Receptor did not show a general trend between experimental biological relative activity and the its stability;• The substitution of Prolines have both a destabilising and stabilising effect in peptide fragment of c-Myc;• Hydrophobic contacts points are the most important kind of interaction between a peptide fragment of c-Myc and the ligand;• Our data also suggest the ligand is bound to the residues located at the middle of the peptide fragment of c-Myc more specifically to the Isoleucine (I49), Tryptophan (W50) and Phenylalanine (F53);• As future perspectives this work might constitute the basis of further evaluation of the ligand as a drug with the possibility to inhibit c-Myc activity, with great importance to for drug discovery.
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