Biology subjects

Marqusee, S.

Publications and source records attributed to Marqusee, S..

31 records · Page 2Linked to original sources

bioRxiv2021

Ligand-induced changes in dynamics mediate long-range allostery in the lac repressor

Allostery, broadly defined as a proteins functional response to distal perturbations, is fundamental to biological regulation. In classical models, allosteric ligand binding produces a defined set of structural changes in the protein, resulting in a different low-energy conformation. Proteins that undergo ligand-induced allostery with few observable structural changes therefore frustrate interpretations by classical models. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map the allosteric effects in a paradigm ligand-responsive allosteric transcription factor, the lac repressor (LacI). X-ray crystal structures of the core domain of LacI bound to different small molecule ligands, or the DNA operator, show less than 1.5 [A] difference in the protein all-atom root-mean-square-deviation (RMSD) between any two structures. Despite this high degree of similarity among static structures, our HDX/MS experiments reveal widespread and unexpected differences in the flexibility of secondary structures in the LacI core domain in each functional state. We propose a model in which ligand binding allosterically switches the functional response of the repressor by selectively changing the dynamics of particular secondary structure elements relative to each other, shifting the conformational ensemble of the protein between mutually incompatible DNA-bound and inducer-bound states. Our model also provides a mechanistic context for the altered functions of thousands of documented LacI mutants. Furthermore, our approach provides a platform for characterizing and engineering allosteric responses in proteins.

biophysics↗

bioRxiv2021

Rigid DNA nanotube tethers suppress high frequency noise in dual-trap optical tweezers systems

Dual trap optical tweezers are a powerful tool to trap and characterize the biophysical properties of single biomolecules such as the folding pathways of proteins and nucleic acids, and the chemomechanical activity of molecular motors. Despite its vastly successful application, noise from drift and fluctuation of the optics, and Brownian motion of the trapped beads still hinder the techniques ability to directly visualize folding of small biomolecules or the single nucleotide stepping of polymerases, especially at low forces (<10 pN) and sub-millisecond timescales. Rigid DNA nanotubes have been used to replace the conventional dsDNA linker to reduce optical tweezers noise in the low force range. However, optical tweezers are used to study a wide range of biophysical events, with timescales ranging from microseconds to seconds, and length changes ranging from sub nanometers to tens of nanometers. In this study, we systematically evaluate how noise is distributed across different frequencies in dual trap optical tweezers systems-and show that rigid DNA nanotube tethers suppress only high frequency noise (kHz), while the low frequency noise remains the same when compared to that of dsDNA tethers.

biophysics↗

bioRxiv2021

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem-SH2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation which stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to doubly-phosphorylated tyrosine-containing motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the auto-inhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

biochemistry↗

bioRxiv2021

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation--an open trimer-- contains easily accessible RBDs. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines. One Sentence SummaryAn alternative conformation of SARS-CoV-2 spike ectodomain modulated by temperature, binding, and sequence variants.

biophysics↗

bioRxiv2020

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity--and, by extension, the myriad effects of ubiquitination on substrate proteins--arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrates C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words) Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a proteins energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate-ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.

biophysics↗

bioRxiv2020

Exploring the evolutionary history of kinetic stability in the alpha-lytic protease family

In addition to encoding the final tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determines the speed of folding. How these kinetic barriers are encoded by the sequence is not well understood. Here, we use evolutionary sequence variation in the alpha-lytic protease (LP) protein family to probe the relationship between sequence and energy landscape. LP has an unusual energy landscape: the native state of LP is not the most thermodynamically favored conformation and, instead, it remains folded due to a large kinetic barrier preventing unfolding. In order to fold, LP utilizes an N-terminal pro region of similar size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant timescale. Without the pro region, LP folds on the order of millennia. A phylogenetic search uncovers LP homologs with a wide range of pro-region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro-region size. Homologs naturally lacking pro regions are thermodynamically stable, fold much faster than LP, yet retain the same fold as LP. Key amino acids thought to contribute to LPs extreme kinetic stability are lost in these homologs, further supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on and changes to the protein sequence.

biophysics↗

bioRxiv2020

The folding and unfolding behavior of ribonuclease H on the ribosome

The health of a cell depends on accurate translation and proper protein folding; misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the energy landscape of the nascent chain. However, quantifying these environmental effects is challenging due to the ribosomal proteins and rRNA, which preclude most spectroscopic measurements of protein energetics. We have applied two gel-based approaches, pulse proteolysis and force-peptide arrest assays, to probe the folding and unfolding pathways of RNase H ribosome-stalled nascent chains. We find that ribosome-stalled RNase H has an increased unfolding rate compared to free RNase H, which completely accounts for observed changes in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNase H on the ribosome. Surprisingly, we find that population of the RNase H folding intermediate is required to generate sufficient force to release the SecM stall and that readthrough of the stall sequence directly correlates with the stability of the folding intermediate. Together, these data imply that the folding pathway of RNase H is unchanged on the ribosome. Furthermore, our data indicate that the ribosome promotes unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of misfolding and assist in folding fidelity.

biophysics↗

bioRxiv2020

Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. Experimental observations of catalysis-enhanced enzyme diffusion, to date, have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an Anti-Brownian ELectrokinetic (ABEL) trap and in-solution spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an Anti-Brownian ELectrokinetic (ABEL) trap and in-solution single-particle tracking (SPT), we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ~20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte-Carlo simulations, we establish that pNPP-induced dye blinking at the ~10 ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution SPT and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale. SIGNIFICANCE STATEMENTRecent experiments have suggested that the energy released by a chemical reaction can propel its enzyme catalyst (for example, alkaline phosphatase, ALP). However, this topic remains controversial, partially due to the indirect and ensemble nature of existing measurements. Here, we used recently developed single-molecule approaches to monitor directly the motions of individual proteins in aqueous solution and find that single ALP enzymes do not diffuse faster under catalysis. Instead, we demonstrate that interactions between the fluorescent dye and the enzymes substrate can produce the signature of apparent diffusion enhancement in fluorescence correlation spectroscopy (FCS), the standard ensemble assay currently used to study enzyme diffusion and indicate that single-molecule approaches provide a more robust means to investigate diffusion at the nanoscale.

biophysics↗

bioRxiv2019

Ubiquitination modulates a protein energy landscape site-specifically with consequences for proteasomal degradation

Cellular environments modulate protein energy landscapes to drive important biology, where small perturbations are consequential for biological signaling, allostery, and other vital processes. The energetic effects of ubiquitination are interesting due to its potential influence on degradation by the 26S proteasome, which requires intrinsically flexible or unstructured initiation regions that many known proteasome substrates lack. We generated proteins with natively attached, isopeptide-linked ubiquitin in structured domains to assess the energetic changes contributed by ubiquitin and how such changes manifest at the proteasome. Ubiquitination at sensitive sites destabilizes the native structure, and thereby increases the rate of degradation for substrates containing unstructured initiation regions. Importantly, this ubiquitination can even induce those requisite regions in well-folded proteins for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.

biochemistry↗

bioRxiv2019

α-carboxysome formation is mediated by the multivalent and disordered protein CsoS2

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism, feeding high concentrations of CO2 to the enzyme Rubisco for fixation. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the -carboxysome, there is little molecular insight into protein-protein interactions which drive the assembly process. Here we show that the N-terminus of CsoS2, an intrinsically disordered protein found in the -carboxysome, possesses a repeated peptide sequence that binds Rubisco. X-ray structural analysis of the peptide bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate CsoS2 acts as an interaction hub to condense Rubisco and enable efficient -carboxysome formation.

biochemistry↗

bioRxiv2018

Modulating long-range energetics via helix stabilization: a case study using T4 lysozyme

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization.

biophysics↗

bioRxiv2018

A small single-domain protein folds through the same pathway on- and off- the ribosome

In vivo, proteins fold and function in a complex environment where they are subject to many stresses that can modulate protein energy landscapes. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemo-mechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein, which appears to fold at the mouth of the ribosome as the protein emerges from the exit tunnel. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multi-domain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.

biophysics↗

bioRxiv2018

Tracing a proteins folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

The conformations populated during protein folding have been studied for decades; yet, their evolutionary importance remains largely unexplored. Ancestral sequence reconstruction allows access to proteins across evolutionary time, and new methods such as pulsed-labeling hydrogen exchange coupled with mass spectrometry allow determination of folding intermediate structures at near amino-acid resolution. Here, we combine these techniques to monitor the folding of the ribonuclease H family along the evolutionary lineages of T. thermophilus and E. coli RNase H. All homologs and ancestral proteins studied populate a similar folding intermediate despite being separated by billions of years of evolution. Even though this conformation is conserved, the pathway leading to it has diverged over evolutionary time, and rational mutations can alter this trajectory. Our results demonstrate that evolutionary processes can affect the energy landscape to preserve or alter specific features of a proteins folding pathway.

biochemistry↗