Publications by authors named "Catherine A Royer"

72 Publications

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture.

Cell Rep 2021 05 27;35(7):109133. Epub 2021 Apr 27.

Department of Chemistry and Chemical Biology, and Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address:

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (M) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the M substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 M protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their M inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PL). HCV drugs that inhibit PL synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir's antiviral activity as much as 10-fold, while those that only inhibit M do not synergize with remdesivir.
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http://dx.doi.org/10.1016/j.celrep.2021.109133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8075848PMC
May 2021

Protein unfolded states populated at high and ambient pressure are similarly compact.

Biophys J 2021 May 4. Epub 2021 May 4.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York. Electronic address:

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.
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http://dx.doi.org/10.1016/j.bpj.2021.04.031DOI Listing
May 2021

The Molecular Basis for Life in Extreme Environments.

Annu Rev Biophys 2021 05 26;50:343-372. Epub 2021 Feb 26.

Department of Chemistry & Chemical Biology, Cornell University, Ithaca, New York 14853, USA.

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understandingits molecular basis in such inhospitable conditions, given that such conditions lead to loss of structure and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments. We present the state of recent progress in extreme environmental genomics. We then present an overview of our current understanding of the biomolecular adaptation to extreme conditions. As our current and future understanding of biomolecular structure-function relationships in extremophiles requires methodologies adapted to extremes of pressure, temperature, and chemical composition, advances in instrumentation for probing biophysical properties under extreme conditions are presented. Finally, we briefly discuss possible future directions in extreme biophysics.
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http://dx.doi.org/10.1146/annurev-biophys-100120-072804DOI Listing
May 2021

An oligomeric switch controls the Mrr-induced SOS response in E. coli.

DNA Repair (Amst) 2021 Jan 6;97:103009. Epub 2020 Nov 6.

Centre de Biochimie Structurale, CNRS, INSERM, Université de Montpellier, 34090, Montpellier, France; Département MICA, INRA, 78350 Jouy-en-Josas, France. Electronic address:

Mrr from Escherichia coli K12 is a type IV restriction endonuclease whose role is to recognize and cleave foreign methylated DNA. Beyond this protective role, Mrr can inflict chromosomal DNA damage that elicits the SOS response in the host cell upon heterologous expression of specific methyltransferases such as M.HhaII, or after exposure to high pressure (HP). Activation of Mrr in response to these perturbations involves an oligomeric switch that dissociates inactive homo-tetramers into active dimers. Here we used scanning number and brightness (sN&B) analysis to determine in vivo the stoichiometry of a constitutively active Mrr mutant predicted to be dimeric and examine other GFP-Mrr mutants compromised in their response to either M.HhaII activity or HP shock. We also observed in vitro the direct pressure-induced tetramer dissociation by HP fluorescence correlation spectroscopy of purified GFP-Mrr. To shed light on the linkages between subunit interactions and activity of Mrr and its variants, we built a structural model of the full-length tetramer bound to DNA. Similar to functionally related endonucleases, the conserved DNA cleavage domain would be sequestered by the DNA recognition domain in the Mrr inactive tetramer, dissociating into an enzymatically active dimer upon interaction with multiple DNA sites.
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http://dx.doi.org/10.1016/j.dnarep.2020.103009DOI Listing
January 2021

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure.

Biophys J 2020 06 23;118(11):2670-2679. Epub 2020 Apr 23.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York. Electronic address:

The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.
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http://dx.doi.org/10.1016/j.bpj.2020.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264842PMC
June 2020

Characterizing proteins in their cellular environment: Examples of recent advances in quantitative fluorescence microscopy.

Protein Sci 2019 07 22;28(7):1210-1221. Epub 2019 May 22.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, 12180.

Quantitative characterization of protein interactions, both intramolecular and intermolecular, is crucial in understanding the mechanisms and regulation of their function. In recent years, it has become possible to obtain such information on protein systems in live cells, from bacteria to mammalian cell lines. This review discusses recent advances in measuring protein folding, absolute concentration, oligomerization, diffusion, transport, and organization at super-resolution.
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http://dx.doi.org/10.1002/pro.3630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6566565PMC
July 2019

Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates.

Biophys J 2019 02 8;116(3):445-453. Epub 2019 Jan 8.

Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York; Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York. Electronic address:

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.
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http://dx.doi.org/10.1016/j.bpj.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369443PMC
February 2019

Exploring Protein Conformational Landscapes Using High-Pressure NMR.

Methods Enzymol 2019 12;614:293-320. Epub 2018 Dec 12.

Centre de Biochimie Structural CNRS Université de Montpellier UMR, Montpellier, France.

Protein conformational landscapes define their functional properties as well as their proteostasis. Hence, detailed mapping of these landscapes is necessary to understand and modulate protein conformation. The combination of high pressure and NMR provides a particularly powerful approach to characterizing protein conformational transitions. First, pressure, because its effects on protein structure arise from elimination of solvent excluded void volume, represents a more subtle perturbation than chemical denaturants, favoring the population of intermediates. Second, the residue-specific and multifaceted nature of NMR observables informs on many local structural properties of proteins, aiding in the characterization of intermediate and excited states.
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http://dx.doi.org/10.1016/bs.mie.2018.07.006DOI Listing
August 2019

Lessons from pressure denaturation of proteins.

J R Soc Interface 2018 10 3;15(147). Epub 2018 Oct 3.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA

Although it is now relatively well understood how sequence defines and impacts global protein stability in specific structural contexts, the question of how sequence modulates the configurational landscape of proteins remains to be defined. Protein configurational equilibria are generally characterized by using various chemical denaturants or by changing temperature or pH. Another thermodynamic parameter which is less often used in such studies is high hydrostatic pressure. This review discusses the basis for pressure effects on protein structure and stability, and describes how the unique mechanisms of pressure-induced unfolding can provide unique insights into protein conformational landscapes.
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http://dx.doi.org/10.1098/rsif.2018.0244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6228469PMC
October 2018

The consequences of cavity creation on the folding landscape of a repeat protein depend upon context.

Proc Natl Acad Sci U S A 2018 08 13;115(35):E8153-E8161. Epub 2018 Aug 13.

Graduate Program in Biochemistry and Biophysics, Rensselaer Polytechnic Institute, Troy, NY 12180;

The effect of introducing internal cavities on protein native structure and global stability has been well documented, but the consequences of these packing defects on folding free-energy landscapes have received less attention. We investigated the effects of cavity creation on the folding landscape of the leucine-rich repeat protein pp32 by high-pressure (HP) and urea-dependent NMR and high-pressure small-angle X-ray scattering (HPSAXS). Despite a modest global energetic perturbation, cavity creation in the N-terminal capping motif (N-cap) resulted in very strong deviation from two-state unfolding behavior. In contrast, introduction of a cavity in the most stable, C-terminal half of pp32 led to highly concerted unfolding, presumably because the decrease in stability by the mutations attenuated the N- to C-terminal stability gradient present in WT pp32. Interestingly, enlarging the central cavity of the protein led to the population under pressure of a distinct intermediate in which the N-cap and repeats 1-4 were nearly completely unfolded, while the fifth repeat and the C-terminal capping motif remained fully folded. Thus, despite modest effects on global stability, introducing internal cavities can have starkly distinct repercussions on the conformational landscape of a protein, depending on their structural and energetic context.
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http://dx.doi.org/10.1073/pnas.1807379115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126725PMC
August 2018

The Deep Sea Osmolyte Trimethylamine N-Oxide and Macromolecular Crowders Rescue the Antiparallel Conformation of the Human Telomeric G-Quadruplex from Urea and Pressure Stress.

Chemistry 2018 Sep 4;24(54):14346-14351. Epub 2018 Sep 4.

Physikalische Chemie I-Biophysikalische Chemie, Fakultät für Chemie und Chemische Biologie, TU Dortmund, Otto-Hahn Str. 4a, 44227, Dortmund, Germany.

Organisms are thriving in the deep sea at pressures up to the 1 kbar level, which imposes severe stress on the conformational dynamics and stability of their biomolecules. The impact of osmolytes and macromolecular crowders, mimicking intracellular conditions, on the effect of pressure on the conformational dynamics of a human telomeric G-quadruplex (G4) DNA is explored in this study employing single-molecule Förster resonance energy transfer (FRET) experiments. In neat buffer, pressurization favors the parallel/hybrid state of the G4-DNA over the antiparallel conformation at ≈400 bar, finally leading to unfolding beyond 1000 bar. High-pressure NMR data support these findings. The folded topological conformers have different solvent accessible surface areas and cavity volumes, leading to different volumetric properties and hence pressure stabilities. The deep-sea osmolyte trimethylamine N-oxide (TMAO) and macromolecular crowding agents are able to effectively rescue the G4-DNA from unfolding in the whole pressure range encountered on Earth.
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http://dx.doi.org/10.1002/chem.201802444DOI Listing
September 2018

Oligomerization of a G protein-coupled receptor in neurons controlled by its structural dynamics.

Sci Rep 2018 Jul 10;8(1):10414. Epub 2018 Jul 10.

IGF, Univ Montpellier, CNRS, INSERM, Montpellier, France.

G protein coupled receptors (GPCRs) play essential roles in intercellular communication. Although reported two decades ago, the assembly of GPCRs into dimer and larger oligomers in their native environment is still a matter of intense debate. Here, using number and brightness analysis of fluorescently labeled receptors in cultured hippocampal neurons, we confirm that the metabotropic glutamate receptor type 2 (mGlu) is a homodimer at expression levels in the physiological range, while heterodimeric GABA receptors form larger complexes. Surprisingly, we observed the formation of larger mGlu oligomers upon both activation and inhibition of the receptor. Stabilizing the receptor in its inactive conformation using biochemical constraints also led to the observation of oligomers. Following our recent observation that mGlu receptors are in constant and rapid equilibrium between several states under basal conditions, we propose that this structural heterogeneity limits receptor oligomerization. Such assemblies are expected to stabilize either the active or the inactive state of the receptor.
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http://dx.doi.org/10.1038/s41598-018-28682-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039492PMC
July 2018

Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer.

J Biol Chem 2018 08 14;293(31):12248-12258. Epub 2018 Jun 14.

Department of Biochemistry, Weill Cornell Medical College, New York, New York 10065. Electronic address:

Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus ) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.
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http://dx.doi.org/10.1074/jbc.RA118.003648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078452PMC
August 2018

G1/S Transcription Factor Copy Number Is a Growth-Dependent Determinant of Cell Cycle Commitment in Yeast.

Cell Syst 2018 05;6(5):539-554.e11

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address:

To understand how commitment to cell division in late G1 phase (Start) is controlled by growth and nutrients in budding yeast, we determined the absolute concentrations of the G1/S transcription factors SBF (composed of Swi4 and Swi6) and MBF (composed of Mbp1 and Swi6), the transcriptional repressor Whi5, and the G1 cyclins, Cln1 and Cln2, in single live yeast cells using scanning number and brightness (sN&B) microscopy. In rich medium, Whi5, Mbp1, and Swi6 concentrations were independent of cell size, whereas Swi4 concentration doubled in G1 phase, leading to a size-dependent decrease in the Whi5/Swi4 ratio. In small cells, SBF and MBF copy numbers were insufficient to saturate target G1/S promoters, but this restriction diminished as cells grew in size. In poor medium, SBF and MBF subunits, as well as Cln1, were elevated, consistent with a smaller cell size at Start. A mathematical model constrained by sN&B data suggested that size- and nutrient-dependent occupancy of G1/S promoters by SBF/MBF helps set the cell size threshold for Start activation.
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http://dx.doi.org/10.1016/j.cels.2018.04.012DOI Listing
May 2018

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

J Mol Biol 2018 04 13;430(9):1336-1349. Epub 2018 Mar 13.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, 12180, NY, USA. Electronic address:

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action.
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http://dx.doi.org/10.1016/j.jmb.2018.03.005DOI Listing
April 2018

Scanning number and brightness yields absolute protein concentrations in live cells: a crucial parameter controlling functional bio-molecular interaction networks.

Biophys Rev 2018 Feb 30;10(1):87-96. Epub 2018 Jan 30.

Program in Biochemistry and Biophysics, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.

Biological function results from properly timed bio-molecular interactions that transduce external or internal signals, resulting in any number of cellular fates, including triggering of cell-state transitions (division, differentiation, transformation, apoptosis), metabolic homeostasis and adjustment to changing physical or nutritional environments, amongst many more. These bio-molecular interactions can be modulated by chemical modifications of proteins, nucleic acids, lipids and other small molecules. They can result in bio-molecular transport from one cellular compartment to the other and often trigger specific enzyme activities involved in bio-molecular synthesis, modification or degradation. Clearly, a mechanistic understanding of any given high level biological function requires a quantitative characterization of the principal bio-molecular interactions involved and how these may change dynamically. Such information can be obtained using fluctation analysis, in particular scanning number and brightness, and used to build and test mechanistic models of the functional network to define which characteristics are the most important for its regulation.
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http://dx.doi.org/10.1007/s12551-017-0394-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803181PMC
February 2018

Monitoring protein folding through high pressure NMR spectroscopy.

Prog Nucl Magn Reson Spectrosc 2017 11 2;102-103:15-31. Epub 2017 Jun 2.

Centre de Biochimie Structural INSERM U1054, CNRS UMMR 5058, Université de Montpellier, Montpellier 34090, France. Electronic address:

High-pressure is a well-known perturbation method used to destabilize globular proteins. It is perfectly reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. In contrast to other perturbation methods such as heat or chemical denaturant that destabilize protein structures uniformly, pressure exerts local effects on regions or domains of a protein containing internal cavities. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility to monitor at a residue level the structural transitions occurring upon unfolding and to determine the kinetic properties of the process. High-pressure NMR experiments can now be routinely performed, owing to the recent development of commercially available high-pressure sample cells. This review summarizes recent advances and some future directions of high-pressure NMR techniques for the characterization at atomic resolution of the energy landscape of protein folding.
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http://dx.doi.org/10.1016/j.pnmrs.2017.05.003DOI Listing
November 2017

Temperature and pressure limits of guanosine monophosphate self-assemblies.

Sci Rep 2017 08 29;7(1):9864. Epub 2017 Aug 29.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Street 4a, 44227, Dortmund, Germany.

Guanosine monophosphate, among the nucleotides, has the unique property to self-associate and form nanoscale cylinders consisting of hydrogen-bonded G-quartet disks, which are stacked on top of one another. Such self-assemblies describe not only the basic structural motif of G-quadruplexes formed by, e.g., telomeric DNA sequences, but are also interesting targets for supramolecular chemistry and nanotechnology. The G-quartet stacks serve as an excellent model to understand the fundamentals of their molecular self-association and to unveil their application spectrum. However, the thermodynamic stability of such self-assemblies over an extended temperature and pressure range is largely unexplored. Here, we report a combined FTIR and NMR study on the temperature and pressure stability of G-quartet stacks formed by disodium guanosine 5'-monophosphate (Na5'-GMP). We found that under abyssal conditions, where temperatures as low as 5 °C and pressures up to 1 kbar are reached, the self-association of Na5'-GMP is most favoured. Beyond those conditions, the G-quartet stacks dissociate laterally into monomer stacks without significantly changing the longitudinal dimension. Among the tested alkali cations, K is the most efficient one to elevate the temperature as well as the pressure limits of GMP self-assembly.
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http://dx.doi.org/10.1038/s41598-017-10689-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574928PMC
August 2017

Putting the Piezolyte Hypothesis under Pressure.

Biophys J 2017 Sep 10;113(5):974-977. Epub 2017 Aug 10.

Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York; Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York. Electronic address:

A group of small molecules that stabilize proteins against high hydrostatic pressure has been classified as piezolytes, a subset of stabilizing cosolutes. This distinction would imply that piezolytes counteract the effects of high hydrostatic pressure through effects on the volumetric properties of the protein. The purpose of this study was to determine if cosolutes proposed to be piezolytes have an effect on the volumetric properties of proteins through direct experimental measurements of volume changes upon unfolding of model proteins lysozyme and ribonuclease A, in solutions containing varying cosolute concentrations. Solutions containing the proposed piezolytes glutamate, sarcosine, and betaine were used, as well as solutions containing the denaturants guanidinium hydrochloride and urea. Changes in thermostability were monitored using differential scanning calorimetry whereas changes in volume were monitored using pressure perturbation calorimetry. Our findings indicate that increasing stabilizing cosolute concentration increases the stability and transition temperature of the protein, but does not change the temperature dependence of volume changes upon unfolding. The results suggest that the pressure stability of a protein in solution is not directly affected by the presence of these proposed piezolytes, and so they cannot be granted this distinction.
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http://dx.doi.org/10.1016/j.bpj.2017.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611670PMC
September 2017

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Biochemistry 2017 05 18;56(21):2715-2722. Epub 2017 May 18.

Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔG°) of -4.4 and -6.9 kcal mol for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔV) was significantly larger for V67 (71 ± 2 mL mol) than for L67 (58 ± 3 mL mol) inteins. The measured difference in ΔV (13 ± 3 mL mol) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.
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http://dx.doi.org/10.1021/acs.biochem.6b01264DOI Listing
May 2017

High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

Nucleic Acids Res 2017 May;45(9):5323-5332

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.
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http://dx.doi.org/10.1093/nar/gkx192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435990PMC
May 2017

NMR and Computation Reveal a Pressure-Sensitive Folded Conformation of Trp-Cage.

J Phys Chem B 2017 02 3;121(6):1258-1267. Epub 2017 Feb 3.

Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York.

Beyond defining the structure and stability of folded states of proteins, primary amino acid sequences determine all of the features of their conformational landscapes. Characterizing how sequence modulates the population of protein excited states or folding pathways requires atomic level detailed structural and energetic information. Such insight is essential for improving protein design strategies, as well as for interpreting protein evolution. Here, high pressure NMR and molecular dynamics simulations were combined to probe the conformational landscape of a small model protein, the tryptophan cage variant, Tc5b. Pressure effects on protein conformation are based on volume differences between states, providing a subtle continuous variable for perturbing conformations. 2D proton TOCSY spectra of Tc5b were acquired as a function of pressure at different temperature, pH, and urea concentration. In contrast to urea and pH which lead to unfolding of Tc5b, pressure resulted in modulation of the structures that are populated within the folded state basin. The results of molecular dynamics simulations on Tc5b displayed remarkable agreement with the NMR data. Principal component analysis identified two structural subensembles in the folded state basin, one of which was strongly destabilized by pressure. The pressure-dependent structural perturbations observed by NMR coincided precisely with the changes in secondary structure associated with the shifting populations in the folded state basin observed in the simulations. These results highlight the deep structural insight afforded by pressure perturbation in conjunction with high resolution experimental and advanced computational tools.
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http://dx.doi.org/10.1021/acs.jpcb.6b11810DOI Listing
February 2017

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

Biophys J 2016 Dec;111(11):2368-2376

Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York. Electronic address:

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific H-N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.
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http://dx.doi.org/10.1016/j.bpj.2016.08.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5153537PMC
December 2016

High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

J Am Chem Soc 2016 11 8;138(46):15260-15266. Epub 2016 Nov 8.

Department of Chemistry & Chemical Biology, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.

Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.
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http://dx.doi.org/10.1021/jacs.6b09887DOI Listing
November 2016

Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability.

DNA Repair (Amst) 2015 Oct 7;34:18-27. Epub 2015 Aug 7.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, United States. Electronic address:

Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag-GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species.
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http://dx.doi.org/10.1016/j.dnarep.2015.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592464PMC
October 2015

Exploring the Protein Folding Pathway with High-Pressure NMR: Steady-State and Kinetics Studies.

Subcell Biochem 2015 ;72:261-78

Centre de Biochimie Structurale, UMR UM1&UM2/5048 CNRS/1054 INSERM, 29 rue de Navacelles, 34090, Montpellier, France.

Defining the physical-chemical determinants of protein folding and stability, under normal and pathological conditions has constituted a major subfield in biophysical chemistry for over 50 years. Although a great deal of progress has been made in recent years towards this goal, a number of important questions remain. These include characterizing the structural, thermodynamic and dynamic properties of the barriers between conformational states on the protein energy landscape, understanding the sequence dependence of folding cooperativity, defining more clearly the role of solvation in controlling protein stability and dynamics and probing the high energy thermodynamic states in the native state basin and their role in misfolding and aggregation. Fundamental to the elucidation of these questions is a complete thermodynamic parameterization of protein folding determinants. In this chapter, we describe the use of high-pressure coupled to Nuclear Magnetic Resonance (NMR) spectroscopy to reveal unprecedented details on the folding energy landscape of proteins.
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http://dx.doi.org/10.1007/978-94-017-9918-8_13DOI Listing
October 2015

Why and How Does Pressure Unfold Proteins?

Subcell Biochem 2015 ;72:59-71

Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA,

This year, 2014, marks the 100th anniversary of the first publication reporting the denaturation of proteins by high hydrostatic pressure (Bridgman 1914). Since that time a large and recently increasing number of studies of pressure effects on protein stability have been published, yet the mechanism for the action of pressure on proteins remains subject to considerable debate. This review will present an overview from this author's perspective of where this debate stands today.
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http://dx.doi.org/10.1007/978-94-017-9918-8_4DOI Listing
October 2015

Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

J Am Chem Soc 2015 Jul 16;137(29):9354-62. Epub 2015 Jul 16.

†Centre de Biochimie Structurale, CNRS UMR5048, INSERM U554, Université Montpellier 1, 29 rue de Navacelles, Montpellier, France 34090.

The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.
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http://dx.doi.org/10.1021/jacs.5b04320DOI Listing
July 2015

Molecular mechanisms of transcriptional control by Rev-erbα: An energetic foundation for reconciling structure and binding with biological function.

Protein Sci 2015 Jul 11;24(7):1129-46. Epub 2015 Jun 11.

Centre de Biochimie Structurale CNRS UMR 5048, INSERM UMR 1054, Université de Montpellier, 34090, Montpellier Cedex, France.

Rev-erbα and β are nuclear receptors that function as transcriptional repressors of genes involved in regulating circadian rhythms, glucose, and cholesterol metabolism and the inflammatory response. Given these key functions, Rev-erbs are important drug targets for treatment of a number of human pathologies, including cancer, heart disease, and type II diabetes. Transcriptional repression by the Rev-erbs involves direct competition with transcriptional activators for target sites, but also recruitment by the Rev-erbs of the NCoR corepressor protein. Interestingly, Rev-erbs do not appear to interact functionally with a very similar corepressor, Smrt. Transcriptional repression by Rev-erbs is thought to occur in response to the binding of heme, although structural, and ligand binding studies in vitro show that heme and corepressor binding are antagonistic. We carried out systematic studies of the ligand and corepressor interactions to address the molecular basis for corepressor specificity and the energetic consequences of ligand binding using a variety of biophysical approaches. Highly quantitative fluorescence anisotropy assays in competition mode revealed that the Rev-erb specificity for the NCoR corepressor lies in the first two residues of the β-strand in Interaction Domain 1 of NCoR. Our studies confirmed and quantitated the strong antagonism of heme and corepressor binding and significant stabilization of the corepressor complex by a synthetic ligand in vitro. We propose a model which reconciles the contradictory observations concerning the effects of heme binding in vitro and in live cells.
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http://dx.doi.org/10.1002/pro.2701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500312PMC
July 2015

The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.

J Cell Sci 2014 Aug 17;127(Pt 16):3451-62. Epub 2014 Jun 17.

CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France INSERM, U661, Montpellier, F-34094, France Universités de Montpellier 1 & 2, UMR-5203, Montpellier, F-34094, France

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.
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http://dx.doi.org/10.1242/jcs.145748DOI Listing
August 2014