Protein Conformational Dynamics Research Articles

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically or into the cross-linker molecules. Isotope-labeled cross-linkers have primarily utilized deuterium and rely on MS1-based quantitation of precursor ion extracted ion chromatograms. Recently the development and application of isobaric quantitative protein interaction reporter (iqPIR) cross-linkers were reported, which utilize 13C and 15N isotope labels. Quantitation is accomplished using relative fragment ion isotope abundances in tandem mass spectra. Here we describe the synthesis and initial evaluation of a multiplexed set of iqPIR molecules, allowing for up to six cross-linked samples to be quantified simultaneously. To analyze data for such cross-linkers, the two-channel mode of iqPIR quantitative analysis was adapted to accommodate any number of channels with defined ion isotope peak mass offsets. The summed ion peak intensities in the overlapping channel isotope envelopes are apportioned among the channels to minimize the difference with respect to the predicted ion isotope envelopes. The result is accurate and reproducible relative quantitation enabling direct comparison among six differentially labeled cross-linked samples. The approach described here is generally extensible for the iqPIR strategy, accommodating future iqPIR reagent design, and enables large-scale in vivo quantitative XL-MS investigation of the interactome.

Single-Molecule Fluorescence Spectroscopy Approaches for Probing Fast Biomolecular Dynamics and Interactions.

Single-molecule fluorescence spectroscopy, and particularly its Förster resonance energy transfer implementation (SM-FRET), provides the opportunity to resolve the stochastic conformational fluctuations undergone by individual protein molecules while they fold-unfold, bind to their partners, or carry out catalysis. Such information is key to resolve the microscopic pathways and mechanisms underlying such processes, and cannot be obtained from bulk experiments. To fully resolve proteinconformational dynamics, SM-FRET experiments need to reach microsecond, and even sub-microsecond, time resolutions. The key to reach such resolution lies in increasing the efficiency at which photons emitted by a single molecule are collected and detected by the instrument (photon count rates). In this chapter, we describe basic procedures that an end user can follow to optimize the confocal microscope optics in order to maximize the photon count rates. We also discuss the use of photoprotection cocktails specifically designed to reduce fluorophore triplet buildup at high irradiance (the major cause of limiting photon emission rates) while improving the mid-term photostability of the fluorophores. Complementary strategies based on the data analysis are discussed in depth by other authors in Chap. 14 .

Hydrogen/Deuterium Exchange Mass Spectrometry with Integrated Electrochemical Reduction and Microchip-Enabled Deglycosylation for Epitope Mapping of Heavily Glycosylated and Disulfide-Bonded Proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a recognized method to study protein conformational dynamics and interactions. Proteins encompassing post-translational modifications (PTMs), such as disulfide bonds and glycosylations, present challenges to HDX-MS, as disulfide bond reduction and deglycosylation is often required to extract HDX information from regions containing these PTMs. In-solution deglycosylation with peptide-N4-(N-acetyl-β-d-glucosaminyl)-asparagine amidase A (PNGase A) or PNGase H+ combined with chemical reduction using tris-(2-carboxyethyl)phosphine (TCEP) has previously been used for HDX-MS analysis of disulfide-linked glycoproteins. However, this workflow requires extensive manual sample preparation and consumes large amounts of enzyme. Furthermore, large amounts of TCEP and glycosidases often result in suboptimal liquid chromatography-mass spectrometry (LC-MS) performance. Here, we compare the in-solution activity of PNGase A, PNGase H+, and the newly discovered PNGase Dj under quench conditions and immobilize them onto thiol-ene microfluidic chips to create HDX-MS-compatible immobilized microfluidic enzyme reactors (IMERs). The IMERS retain deglycosylation activity, also following repeated use and long-term storage. Furthermore, we combine a PNGase Dj IMER, a pepsin IMER, and an electrochemical cell to develop an HDX-MS setup capable of efficient online disulfide-bond reduction, deglycosylation, and proteolysis. We demonstrate the applicability of this setup by mapping the epitope of a monoclonal antibody (mAb) on the heavily disulfide-bonded and glycosylated sema-domain of the tyrosine-protein kinase Met (SD c-Met). We achieve near-complete sequence coverage and extract HDX data to identify regions of SD c-Met involved in mAb binding. The described methodology thus presents an integrated and online workflow for improved HDX-MS analysis of challenging PTM-rich proteins.

Protein conformational dynamics and phenotypic switching.

Intrinsically disordered proteins (IDPs) are proteins that lack rigid 3D structure but exist as conformational ensembles. Because of their structural plasticity, they can interact with multiple partners. The protein interactions between IDPs and their partners form scale-free protein interaction networks (PINs) that facilitate information flow in the cell. Because of their plasticity, IDPs typically occupy hub positions in cellular PINs. Furthermore, their conformational dynamics and propensity for post-translational modifications contribute to "conformational" noise which is distinct from the well-recognized transcriptional noise. Therefore, upregulation of IDPs in response to a specific input, such as stress, contributes to increased noise and, hence, an increase in stochastic, "promiscuous" interactions. These interactions lead to activation of latent pathways or can induce "rewiring" of the PIN to yield an optimal output underscoring the critical role of IDPs in regulating information flow. We have used PAGE4, a highly intrinsically disordered stress-response protein as a paradigm. Employing a variety of experimental and computational techniques, we have elucidated the role of PAGE4 in phenotypic switching of prostate cancer cells at a systems level. These cumulative studies over the past decade provide a conceptual framework to better understand how IDP conformational dynamics and conformational noise might facilitate cellular decision-making.

PTMdyna: exploring the influence of post-translation modifications on protein conformational dynamics.

Protein post-translational modifications (PTM) play vital roles in cellular regulation, modulating functions by driving changes in protein structure and dynamics. Exploring comprehensively the influence of PTM on conformational dynamics can facilitate the understanding of the related biological function and molecular mechanism. Currently, a series of excellent computation tools have been designed to analyze the time-dependent structural properties of proteins. However, the protocol aimed to explore conformational dynamics of post-translational modified protein is still a blank. To fill this gap, we present PTMdyna to visually predict the conformational dynamics differences between unmodified and modified proteins, thus indicating the influence of specific PTM. PTMdyna exhibits an AUC of 0.884 tested on 220 protein-protein complex structures. The case of heterochromatin protein 1α complexed with lysine 9-methylated histone H3, which is critical for genomic stability and cell differentiation, was used to demonstrate its applicability. PTMdyna provides a reliable platform to predict the influence of PTM on protein dynamics, making it easier to interpret PTM functionality at the structure level. The web server is freely available at http://ccbportal.com/PTMdyna.

Conformational changes and binding property of the periplasmic binding protein BtuF during vitamin B12 transport revealed by collision-induced unfolding, hydrogen-deuterium exchange mass spectrometry and molecular dynamic simulation

The periplasmic binding protein (PBP) BtuF plays a key role in transporting vitamin B12 from periplasm to the ATP-binding cassette (ABC) transporter BtuCD. Conformational changes of BtuF during transport can hardly be captured by traditional biophysical methods and the exact mechanism regarding B12 and BtuF recognition is still under debate. In the present work, conformational changes of BtuF upon B12 binding and release were investigated using hybrid approaches including collision-induced unfolding (CIU), hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulation. It was found that B12 binding increased the stability of BtuF. In addition, fast exchange regions of BtuF were localized. Most importantly, midpoint of hinge helix in BtuF was found highly flexible, and binding of B12 proceed in a manner similar to the Venus flytrap mechanism. Our study therefore delineates a clear view of BtuF delivering B12, and demonstrated a hybrid approach encompassing MS and computer based methods that holds great potential to the probing of conformational dynamics of proteins in action.

Correlation of membrane protein conformational and functional dynamics

Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

Ultra-sensitive lock-in amplifier coupled oscillatory magnetic tweezers for piconewton force manipulation applications

We have developed lock-in amplifier coupled oscillatory magnetic tweezers, aiming to synchronize the oscillatory magnetic force application and single-molecule response detection at the applied oscillatory frequency by incorporating an optical lock-in amplifier detection. The designed home-built lock-in amplifier detection instrument enables an exact reference signal input and hence reveals an accurate and sensitive magnetic response synchronization. We have further demonstrated the approach with the rhodamine 6G stained super-paramagnetic beads by monitoring the below-the-noise-background weak fluorescence signal changes due to the magnetic response of the super-paramagnetic beads under the oscillatory force manipulation. The integration of the lock-in amplifier and the oscillating magnetic tweezers can significantly expand the application of the magnetic tweezers for signal detection below the noise background, such as adapting to the important applications in the detailed exploration of mechanical properties of biomolecules and studies of the protein conformational fluctuation dynamics.

Interactive Interface for Graph-Based Analyses of Dynamic H-Bond Networks: Application to Spike Protein S.

Dynamic hydrogen-bond networks are key determinants of protein conformational dynamics. In the case of macromolecular protein complexes, which can have a large number of hydrogen bonds giving rise to extensive hydrogen-bond networks, efficient algorithms are required to analyze interactions that could be important for the dynamics and biological function of the complex. We present here a highly efficient, standalone interface designed for analyses of dynamical hydrogen-bond networks of biomolecules and macromolecular complexes. To facilitate a comprehensive description of protein dynamics, the interface includes analyses of hydrophobic interactions. We illustrate the usefulness and workflow of the interface by dissecting the dynamics of the ectodomain of SARS-CoV-2 protein S in its closed conformation. We find that protein S contains numerous local clusters of dynamic hydrogen bonds and identify hydrogen bonds that are sampled persistently. The receptor binding domain of the spike protein hosts only a handful of persistent hydrogen-bond clusters, suggesting structural plasticity. Our data analysis interface is released here for open use.

Multiscale Simulations on the Catalytic Plasticity of CYP76AH1.

The catalytic promiscuity and fidelity of cytochrome P450 enzymes are widespread in the skeletal modification of terpenoid natural products and have attracted much attention. CYP76AH1 is involved in key modification reactions in the biosynthetic pathway of tanshinone, a well-known medicinal norditerpenoid. In this work, classical molecular dynamic simulations, metadynamics, and DFT calculations were performed to investigate the protein conformational dynamics, ligand binding poses, and catalytic reaction mechanism in wide-type and mutant CYP76AH1. Our results not only reveal a plausible enzymatic mechanism for mutant CYP76AH1 leading to various products but also provide valuable guidance for rational protein engineering of the CYP76 family.

Abstract P-10: Solvatochromic Fluorescent Dyes Tested for Spectroscopic Measurements of Protein Conformational Dynamics

Background: Recoverin is a 23 kDa protein, belonging to the superfamily of EF-hand Ca2+-binding proteins. One of the functions of recoverin is to regulate the activity of the rhodopsin kinase GRK1, which regulates the activity of rhodopsin. In dim ambient light, the level of calcium in the rod cells of the retina is high, so recoverin binds to and inhibits rhodopsin kinase, leaving rhodopsin very sensitive to photons to enable the eye to detect visual signals even under low-light conditions. Many biophysical methods have previously been used to study the conformational dynamics of recoverin, including NMR, SPR and fluorescence spectroscopy. Here we describe fluorescent solvatochromic dyes suitable for spectroscopic observation of conformational changes in recoverin. Methods: We tested four fluorescent dyes, which were covalently attached to Cys39 of recoverin via the thiol-maleimide interaction. Results: Two out of four labeled recoverin samples showed EGTA-induced changes in the fluorescence lifetime and excitation and emission spectra. Conclusion: Our experiments show solvatochromic fluorescent dyes that can be successfully used for spectroscopic observation of conformational dynamics in proteins.

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins.

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

Direct Observation of MAX1 Self‐Assembly via Site‐Specific Carbon‐Deuterium Infrared Probes

The physical characterization of molecules that self-assemble presents a challenge, particularly when associated with phase separation or precipitation. Infrared (IR) spectroscopy has been shown to be a non-perturbative method for monitoring folding dynamics, accentuated by the sensitivity of the bond stretch frequencies of site-specific IR probes to changes in local molecular environment.1 Site-specific incorporation of probes that absorb in the “transparent window” region of the biomolecular IR spectrum has been widely used to gain insight into protein electrostatics, structure, folding, and conformational dynamics. To expand the use of IR probes for the investigation of time-resolved kinetic processes, such as self-assembly, carbon-deuterium (C-D) bonds were incorporated into five different positions of a twenty-residue MAX1 β-hairpin peptide and the site-specific kinetics of gelation were examined after stopped-flow mixing using FTIR detection. MAX1 peptide hydrogels are biocompatible and clinically relevant for application in tissue regeneration and drug delivery.2 Steady state IR absorption frequencies and linewidths of C-D bonds at all labeled positions indicate, as expected, that the labeled side chains occupy a hydrophobic region of the hydrogel; furthermore, the motion of side chains located in the middle of the hairpin is more restricted than those located on the hairpin ends. The changes in the absorption spectra of C-D bonds induced by gelation were reliably followed as function of time and the site-specific time constants for the gel transition show that MAX1 gelation occurs as a cooperative process with no lag phase. We believe that stopped-flow FTIR spectroscopy is equally applicable for other transparent window IR probes and can be extended to other time-resolved applications, such as protein folding and enzyme kinetics. 1. Adhikary, R., J. Zimmermann, and F.E. Romesberg, Transparent Window Vibrational Probes for the Characterization of Proteins With High Structural and Temporal Resolution. Chem Rev, 2017. 117(3): p. 1927-1969. 2. Schneider, J.P., et al., Responsive Hydrogels from the intramolecular Folding and Self-Assembly of a Designed Peptide. J. of Am. Chem. Soc., 2002. 124: p. 15030-15037.

Origin and Control of Chemoselectivity in Cytochrome c Catalyzed Carbene Transfer into Si-H and N-H bonds.

A cytochrome c heme protein was recently engineered to catalyze the formation of carbon-silicon bonds via carbene insertion into Si-H bonds, a reaction that was not previously known to be catalyzed by a protein. High chemoselectivity toward C-Si bond formation over competing C-N bond formation was achieved, although this trait was not screened for during directed evolution. Using computational and experimental tools, we now establish that activity and chemoselectivity are modulated by conformational dynamics of a protein loop that covers the substrate access to the iron-carbene active species. Mutagenesis of residues computationally predicted to control the loop conformation altered the protein's chemoselectivity from preferred silylation to preferred amination of a substrate containing both N-H and Si-H functionalities. We demonstrate that information on protein structure and conformational dynamics, combined with knowledge of mechanism, leads to understanding of how non-natural and selective chemical transformations can be introduced into the biological world.

What Markov State Models Can and Cannot Do: Correlation versus Path-Based Observables in Protein-Folding Models.

Markov state models (MSMs) have been widely applied to study the kinetics and pathways of protein conformational dynamics based on statistical analysis of molecular dynamics (MD) simulations. These MSMs coarse-grain both configuration space and time in ways that limit what kinds of observables they can reproduce with high fidelity over different spatial and temporal resolutions. Despite their popularity, there is still limited understanding of which biophysical observables can be computed from these MSMs in a robust and unbiased manner, and which suffer from the space-time coarse-graining intrinsic in the MSM model. Most theoretical arguments and practical validity tests for MSMs rely on long-time equilibrium kinetics, such as the slowest relaxation time scales and experimentally observable time-correlation functions. Here, we perform an extensive assessment of the ability of well-validated protein folding MSMs to accurately reproduce path-based observable such as mean first-passage times (MFPTs) and transition path mechanisms compared to a direct trajectory analysis. We also assess a recently proposed class of history-augmented MSMs (haMSMs) that exploit additional information not accounted for in standard MSMs. We conclude with some practical guidance on the use of MSMs to study various problems in conformational dynamics of biomolecules. In brief, MSMs can accurately reproduce correlation functions slower than the lag time, but path-based observables can only be reliably reproduced if the lifetimes of states exceed the lag time, which is a much stricter requirement. Even in the presence of short-lived states, we find that haMSMs reproduce path-based observables more reliably.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the µs-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the µs-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers.

Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand-receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics - from protein synthesis to physiological function - as well as emerging future issues that are beginning to be addressed with novel approaches.

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization "Hotspots" in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction.

The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these “binding-site adjacent” dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these “peripheral” sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization “hotspots” outside of the core binding interfaces defined by high resolution structural data.

Molecular Fluctuations as a Ruler of Force-Induced Protein Conformations.

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis examines protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein’s end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein’s contour length, in agreement with the sequence-independent freely-jointed chain model of polymer physics. Our findings will help elucidate the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.