Hydroxyl Radical Footprinting Research Articles

Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells.

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria that encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semipermeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We used molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed the overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

REVIEW ON PROTEIN FOOTPRINTING AS A TOOL IN STRUCTURAL BIOLOGY

Structural characterization of proteins is fundamental for understanding their functions and interactions within cellular environments. Protein footprinting has emerged as a powerful and versatile technique in structural biology, providing valuable insights into protein structure, dynamics, and conformational changes. This study comprehensively explores the principles, methodologies, and applications of protein footprinting as an indispensable tool in structural biology. Protein footprinting encompasses a range of innovative techniques, including hydroxyl radical footprinting, mass spectrometry-based footprinting, and chemical footprinting, each offering unique advantages for probing different aspects of protein structure. The study elucidates the underlying principles of these methods, discussing the mechanisms by which protein-footprinting agents interact with amino acid residues and elucidate structural details. The application of protein footprinting in studying protein-protein interactions, ligand binding, and conformational changes is highlighted through a detailed examination of key studies and experimental approaches. The review emphasizes the versatility of protein footprinting in investigating diverse protein systems, from small globular proteins to large multi-subunit complexes. Furthermore, the integration of protein footprinting with other structural biology techniques, such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy, is explored to showcase the complementary nature of these approaches in achieving a comprehensive understanding of protein structure and function. The study addresses recent advancements and technological innovations in protein footprinting methodologies, including advancements in mass spectrometry instrumentation and data analysis strategies. Additionally, it discusses challenges associated with the technique, such as data interpretation and accessibility, and proposes future directions for overcoming these hurdles. Protein footprinting stands as a valuable tool in the structural biologist’s arsenal, providing unique perspectives on protein structure and dynamics. As the field continues to evolve, with ongoing refinements in methodologies and expanded applications, protein footprinting is poised to play a pivotal role in unraveling the complexities of biological macromolecules. This study serves as a comprehensive resource for researchers and practitioners seeking an in-depth understanding of protein footprinting and its contributions to advancing structural biology.

Structural Analysis of Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using Flash Oxidation Coupled In-Line with Ion Exchange Chromatography.

Protein posttranslational modifications (PTMs) are key modulators of protein structure and function that often change in a dynamic fashion in response to cellular stimuli. Dynamic PTMs are very challenging to structurally characterize using modern techniques, including covalent labeling methods, due to the presence of multiple proteoforms and conformers together in solution. We have coupled an ion exchange high-performance liquid chromatography separation with a flash oxidation system [ion exchange chromatography liquid chromatography-flash oxidation (IEX LC-FOX)] to successfully elucidate structural changes among three phosphoproteoforms of ovalbumin (OVA) during dephosphorylation with alkaline phosphatase. Real-time dosimetry indicates no difference in the effective radical dose between peaks or across the peak, demonstrating both the lack of scavenging of the NaCl gradient and the lack of a concentration effect on radical dose between peaks of different intensities. The use of IEX LC-FOX allows us to structurally probe into each phosphoproteoform as it elutes from the column, capturing structural data before the dynamics of the system to reintroduce heterogeneity. We found significant differences in the residue-level oxidation between the hydroxyl radical footprint of nonphosphorylated, monophosphorylated, and diphosphorylated OVA. Not only were our data consistent with the previously reported stabilization of OVA structure by phosphorylation, but local structural changes were also consistent with the measured order of dephosphorylation of Ser344 being removed first. These results demonstrate the utility of IEX LC-FOX for measuring the structural effects of PTMs, even in dynamic systems.

Structural Investigation of Therapeutic Antibodies Using Hydroxyl Radical Protein Footprinting Methods.

Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to refine antibody design. However, many standard structural techniques require specialized sample preparation that may perturb antibody structure or require high concentrations or other conditions that are far from the conditions conducive to the accurate determination of antigen binding or kinetics. We describe here in this minireview the relatively new method of hydroxyl radical protein footprinting, a solution-state method that can provide structural and kinetic information on antibodies or antibody-antigen interactions useful for therapeutic antibody design. We provide a brief history of hydroxyl radical footprinting, examples of current implementations, and recent advances in throughput and accessibility.

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation.

Mass spectrometry-based analyses of protein conformation continue to grow in utilization due their speed, low sample requirements, and applicability to most protein systems. These techniques typically rely on chemical derivatization of proteins and as with all label-based analyses must ensure the integrity of the protein conformation throughout the duration of the labeling reaction. Hydroxyl radical footprinting of proteins and the recently developed fast fluoroalkylation of proteins attempt to bypass this consideration via rapid reactions that occur on time scales faster than protein folding, but they often require microfluidic setups or electromagnetic radiation sources. In this work, we demonstrate that ozonation of proteins and peptides, which normally occurs in the second to minute time scales, can be accelerated to the submillisecond to millisecond time scale with an electrospray ionization source. This rapid ozonation results in selective labeling of tryptophan and methionine residues. When applied to cytochrome C and carbonic anhydrase, this labeling technique is sensitive to solution conditions and correlates with solution-phase analyses of conformation. While significant work is still needed to characterize this fast chemical labeling strategy, it requires no complicated sample handling, electromagnetic radiation sources, or microfluidic systems outside of the electrospray source and may represent a facile alternative to other rapid labeling technologies that are utilized today.

Structural basis for p53 binding to its nucleosomal target DNA sequence.

The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its target DNA in the nucleosome remains elusive. Here we report the cryo-electron microscopy structures of the p53 DNA-binding domain and the full-length p53 protein complexed with a nucleosome containing the 20 base-pair target DNA sequence of p53 (p53BS). In the p53-nucleosome structures, the p53 DNA-binding domain forms a tetramer and specifically binds to the p53BS DNA, located near the entry/exit region of the nucleosome. The nucleosomal position of the p53BS DNA is within the genomic p21 promoter region. The p53 binding peels the DNA from the histone surface, and drastically changes the DNA path around the p53BS on the nucleosome. The C-terminal domain of p53 also binds to the DNA around the center and linker DNA regions of the nucleosome, as revealed by hydroxyl radical footprinting. These results provide important structural information for understanding the mechanism by which p53 binds the nucleosome and changes the chromatin structure for gene activation.

Fast photochemical oxidation of proteins coupled with mass spectrometry

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes. FPOP has been applied to structurally diverse and biopharmaceutically relevant systems from small, monomeric aggregation-prone proteins to proteome-wide analysis of whole organisms. This review evaluates the current state of FPOP, the progress needed to address data analysis bottlenecks, particularly for residue-level analysis, and highlights significant developments of the FPOP platform that have enabled its versatility and complementarity to other structural biology techniques.

Integration of Mass Spectrometry Data for Structural Biology.

Mass spectrometry (MS) is increasingly being used to probe the structure and dynamics of proteins and the complexes they form with other macromolecules. There are now several specialized MS methods, each with unique sample preparation, data acquisition, and data processing protocols. Collectively, these methods are referred to as structural MS and include cross-linking, hydrogen-deuterium exchange, hydroxyl radical footprinting, native, ion mobility, and top-down MS. Each of these provides a unique type of structural information, ranging from composition and stoichiometry through to residue level proximity and solvent accessibility. Structural MS has proved particularly beneficial in studying protein classes for which analysis by classic structural biology techniques proves challenging such as glycosylated or intrinsically disordered proteins. To capture the structural details for a particular system, especially larger multiprotein complexes, more than one structural MS method with other structural and biophysical techniques is often required. Key to integrating these diverse data are computational strategies and software solutions to facilitate this process. We provide a background to the structural MS methods and briefly summarize other structural methods and how these are combined with MS. We then describe current state of the art approaches for the integration of structural MS data for structural biology. We quantify how often these methods are used together and provide examples where such combinations have been fruitful. To illustrate the power of integrative approaches, we discuss progress in solving the structures of the proteasome and the nuclear pore complex. We also discuss how information from structural MS, particularly pertaining to protein dynamics, is not currently utilized in integrative workflows and how such information can provide a more accurate picture of the systems studied. We conclude by discussing new developments in the MS and computational fields that will further enable in-cell structural studies.

Structurally distinct external solvent-exposed domains drive replication of major human prions.

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques—mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures.

Antibody–receptor interactions mediate antibody-dependent cellular cytotoxicity

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody–receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting–MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab–receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab–receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc–polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG–Fc receptor biology.

Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

Abstract A48: Therapeutic reactivation of the protein phosphatase 2A (PP2A) for the treatment of KRAS-driven cancers

Abstract There have been tremendous strides in the development of therapies for the treatment of NSCLC over the past decade. Nevertheless, the most common recurrent mutation driving the growth of NSCLC, mutant KRAS, accounting for ~25% of patients with advanced NSCLC, remains without an effective therapy. Thus, novel therapies are critically needed to improve the lives of patients suffering from KRAS-driven lung cancers. Protein phosphatase 2A (PP2A) is a serine threonine-directed tumor-suppressive phosphatase that is dysregulated and deactivated in cancer by multiple mechanisms including somatic mutation, suppression of individual subunits, increased expression of endogenous PP2A inhibitors and changes in post-translational modifications to the catalytic C subunit. Given its central role in regulating many key cellular processes and its role in human disease pathogenesis, many efforts have been developed to therapeutically target PP2A. Our group has developed first-in-class direct small-molecule activators of PP2A (SMAPs) by using the chemical scaffold of the FDA-approved tricyclic neuroleptics. We have used molecular modeling, hydroxyl radical footprinting and cryo-electron microscopy to structurally resolve the mechanism of action of SMAP-mediated growth suppression. Specifically, SMAPs protect the regulatory c-terminal tail of the catalytic subunit through changes in the methylation of the terminal leucine (L309) that induces the holoenzyme heterotrimerization and increased substrate-directed catalysis. SMAP treatment of KRAS-mutant lung cancer cells induced apoptosis and decreased the phosphorylation of PP2A targets including AKT and ERK. We found that treatment of xenograft, PDX, and GEMM mouse models of KRAS-mutant lung cancer cells with SMAPs resulted in marked tumor growth inhibition. Taken together, these studies demonstrate the potential of using SMAPs as an approach to treat KRAS-mutant NSCLC. We also established that PP2A activity defines the response of KRAS-mutant lung cancer cells across library of over 200 kinase inhibitors. In vivo studies using xenograft mouse models of KRAS-mutant lung adenocarcinoma demonstrated that SMAP in combination with MEK inhibitor resulted in tumor regression. Our studies have identified a combination of drugs that is effective in vivo for the treatment of KRAS-mutant lung cancer. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other KRAS-driven cancers and represents a first step into a new territory of developing small-molecule activators of tumor-suppressor proteins. Citation Format: Jaya Sangodkar, Jukka Westermarck, Derek Taylor, Goutham Narla. Therapeutic reactivation of the protein phosphatase 2A (PP2A) for the treatment of KRAS-driven cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A48.

Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain with a Small Molecule Inhibitor.

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length

Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein-protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.

Water molecules mediate zinc mobility in the bacterial zinc diffusion channel ZIPB

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.

Illuminating Biological Interactions with in Vivo Protein Footprinting.

Protein footprinting coupled with mass spectrometry is being increasingly used for the study of protein interactions and conformations. The hydroxyl radical footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to oxidatively modify solvent accessible amino acids. Here, we describe the further development of FPOP for protein structural analysis in vivo (IV-FPOP) with Caenorhabditis elegans. C. elegans, part of the nematode family, are used as model systems for many human diseases. The ability to perform structural studies in these worms would provide insight into the role of structure in disease pathogenesis. Many parameters were optimized for labeling within the worms including the microfluidic flow system and hydrogen peroxide concentration. IV-FPOP was able to modify several hundred proteins in various organs within the worms. The method successfully probed solvent accessibility similarily to in vitro FPOP, demonstrating its potential for use as a structural technique in a multiorgan system. The coupling of the method with mass spectrometry allows for amino-acid-residue-level structural information, a higher resolution than currently available in vivo methods.

Complex between a Multicrossover DNA Nanostructure, PX-DNA, and T7 Endonuclease I.

Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although existing evidence has suggested that PX is the DNA motif in homologous pairing (HP), this conclusion remains ambiguous. Further investigation is needed but will require development of new tools. Here, we report characterization of the complex between PX-DNA and T7 endonuclease I (T7endoI), a junction-resolving protein that could serve as the prototype of an anti-PX ligand (a critical prerequisite for the future development of such tools). Specifically, nuclease-inactive T7endoI was produced and its ability to bind to PX-DNA was analyzed using a gel retardation assay. The molar ratio of PX to T7endoI was determined using gel electrophoresis and confirmed by the Hill equation. Hydroxyl radical footprinting of T7endoI on PX-DNA is used to verify the positive interaction between PX and T7endoI and to provide insight into the binding region. Cleavage of PX-DNA by wild-type T7endoI produces DNA fragments, which were used to identify the interacting sites on PX for T7endoI and led to a computational model of their interaction. Altogether, this study has identified a stable complex of PX-DNA and T7endoI and lays the foundation for engineering an anti-PX ligand, which can potentially assist in the study of molecular mechanisms for HP at an advanced level.

Recent Advances in X-Ray Hydroxyl Radical Footprinting at the Advanced Light Source Synchrotron

Synchrotron hydroxyl radical footprinting is a relatively new structural method used to investigate structural features and conformational changes of nucleic acids and proteins in the solution state. It was originally developed at the National Synchrotron Light Source at Brookhaven National Laboratory in the late nineties, and more recently, has been established at the Advanced Light Source at Lawrence Berkeley National Laboratory. The instrumentation for this method is an active area of development, and includes methods to increase dose to the samples while implementing high-throughput sample delivery methods. Improving instrumentation to irradiate biological samples in real time using a sample droplet generator and inline fluorescence monitoring to rapidly determine dose response curves for samples will significantly increase the range of biological problems that can be investigated using synchrotron hydroxyl radical footprinting.

Current Trends in Biotherapeutic Higher Order Structure Characterization by Irreversible Covalent Footprinting Mass Spectrometry.

Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development. Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.

Nucleotide Excision Repair and Impact of Site-Specific 5',8-Cyclopurine and Bulky DNA Lesions on the Physical Properties of Nucleosomes.

The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N2-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs but are good substrates of NER in HeLa-NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs, the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa-NCPs, the Förster resonance energy transfer values, or hydroxyl radical footprint patterns and is weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes (≥30 bp). These observations are consistent with the hypothesis that post-translational modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.