Publications by authors named "Osman Bilsel"

37 Publications

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Proc Natl Acad Sci U S A 2021 Jun;118(23)

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605;

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.
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http://dx.doi.org/10.1073/pnas.2024605118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201830PMC
June 2021

Heterogeneous folding and stretched kinetics.

Authors:
Osman Bilsel

Proc Natl Acad Sci U S A 2020 08 28;117(32):18915-18917. Epub 2020 Jul 28.

Microgradient Fluidics LLC, Worcester, MA 01605;

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http://dx.doi.org/10.1073/pnas.2009596117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431018PMC
August 2020

Friction-Limited Folding of Disulfide-Reduced Monomeric SOD1.

Biophys J 2020 04 12;118(8):1992-2000. Epub 2020 Mar 12.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts. Electronic address:

The folding reaction of a stable monomeric variant of Cu/Zn superoxide dismutase (mSOD1), an enzyme responsible for the conversion of superoxide free radicals into hydrogen peroxide and oxygen, is known to be among the slowest folding processes that adhere to two-state behavior. The long lifetime, ∼10 s, of the unfolded state presents ample opportunities for the polypeptide chain to transiently sample nonnative structures before the formation of the productive folding transition state. We recently observed the formation of a nonnative structure in a peptide model of the C-terminus of SOD1, a sequence that might serve as a potential source of internal chain friction-limited folding. To test for friction-limited folding, we performed a comprehensive thermodynamic and kinetic analysis of the folding mechanism of mSOD1 in the presence of the viscogens glycerol and glucose. Using a, to our knowledge, novel analysis of the folding reactions, we found the disulfide-reduced form of the protein that exposes the C-terminal sequence, but not its disulfide-oxidized counterpart that protects it, experiences internal chain friction during folding. The sensitivity of the internal friction to the disulfide bond status suggests that one or both of the cross-linked regions play a critical role in driving the friction-limited folding. We speculate that the molecular mechanisms giving rise to the internal friction of disulfide-reduced mSOD1 might play a role in the amyotrophic lateral sclerosis-linked aggregation of SOD1.
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http://dx.doi.org/10.1016/j.bpj.2020.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175461PMC
April 2020

Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.

Structure 2019 12 7;27(12):1782-1797.e7. Epub 2019 Oct 7.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address:

Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.
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http://dx.doi.org/10.1016/j.str.2019.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948192PMC
December 2019

In Situ Measurements of Polymer Micellization Kinetics with Millisecond Temporal Resolution.

Macromolecules 2019 May 15;52(9):3151-3157. Epub 2019 Apr 15.

Department of Chemical Engineering, University of Illinois at Chicago, Chicago, IL 60607, United States.

Utilizing synchrotron small-angle X-ray scattering (SAXS) integrated with a microfluidic device, micellization kinetics of a diblock co-polymer, poly(ethylene glycol)--poly(caprolactone) (PEG--PCL) was measured in situ with millisecond temporal and micrometer spatial resolution. The evolutionary regimes of polymer micellization - nucleation, fusion, and insertion were directly observed. The five-inlet microfluidic device provided steady continuous mixing of the polymer solution and the antisolvent. Solvent replacement was mainly dominated by lateral diffusion across the hydrodynamically focused central layer, whose thickness could be precisely designed and manipulated from mass balance of the partitioning streams. Knowing the micellization kinetics of the polymers is essential for design and optimization of self-assembled polymeric nanostructures. The technique of integrating SAXS with microfluidic devices can be translatable to other systems for a breadth of applications.
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http://dx.doi.org/10.1021/acs.macromol.8b02257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777845PMC
May 2019

Frustration and folding of a TIM barrel protein.

Proc Natl Acad Sci U S A 2019 08 25;116(33):16378-16383. Epub 2019 Jul 25.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605;

Triosephosphate isomerase (TIM) barrel proteins have not only a conserved architecture that supports a myriad of enzymatic functions, but also a conserved folding mechanism that involves on- and off-pathway intermediates. Although experiments have proven to be invaluable in defining the folding free-energy surface, they provide only a limited understanding of the structures of the partially folded states that appear during folding. Coarse-grained simulations employing native centric models are capable of sampling the entire energy landscape of TIM barrels and offer the possibility of a molecular-level understanding of the readout from sequence to structure. We have combined sequence-sensitive native centric simulations with small-angle X-ray scattering and time-resolved Förster resonance energy transfer to monitor the formation of structure in an intermediate in the indole-3-glycerol phosphate synthase TIM barrel that appears within 50 μs and must at least partially unfold to achieve productive folding. Simulations reveal the presence of a major and 2 minor folding channels not detected in experiments. Frustration in folding, i.e., backtracking in native contacts, is observed in the major channel at the initial stage of folding, as well as late in folding in a minor channel before the appearance of the native conformation. Similarities in global and pairwise dimensions of the early intermediate, the formation of structure in the central region that spreads progressively toward each terminus, and a similar rate-limiting step in the closing of the β-barrel underscore the value of combining simulation and experiment to unravel complex folding mechanisms at the molecular level.
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http://dx.doi.org/10.1073/pnas.1900880116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697809PMC
August 2019

Nonnative structure in a peptide model of the unfolded state of superoxide dismutase 1 (SOD1): Implications for ALS-linked aggregation.

J Biol Chem 2019 09 24;294(37):13708-13717. Epub 2019 Jul 24.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding free-energy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the local behavior of the corresponding sequences in the unfolded ensemble. Using time-resolved FRET, we observed that the peptide corresponding to the Loop VII-β8 sequence at the SOD1 C terminus was uniquely sensitive to denaturant. Utilizing a two-dimensional form of maximum entropy modeling, we demonstrate that the sensitivity to denaturant is the surprising result of a two-state-like transition from a compact to an expanded state. Variations of the peptide sequence revealed that the compact state involves a nonnative interaction between the disordered N terminus and the hydrophobic C terminus of the peptide. This nonnative intramolecular structure could serve as a precursor for intermolecular association and result in aggregation associated with ALS. We propose that this precursor would provide a common molecular target for therapeutic intervention in the dozens of ALS-linked SOD1 mutations.
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http://dx.doi.org/10.1074/jbc.RA119.008765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746449PMC
September 2019

Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions.

Proc Natl Acad Sci U S A 2019 06 5;116(25):12301-12310. Epub 2019 Jun 5.

Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400;

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.
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http://dx.doi.org/10.1073/pnas.1818206116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056937PMC
June 2019

Transient Kinetic Analysis of SWR1C-Catalyzed H2A.Z Deposition Unravels the Impact of Nucleosome Dynamics and the Asymmetry of Histone Exchange.

Cell Rep 2019 04;27(2):374-386.e4

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

The SWR1C chromatin remodeling enzyme catalyzes ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes such as transcription and DNA repair. Here, we investigate the transient kinetic mechanism of the histone exchange reaction, employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady-state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA unwrapping. Taken together, our transient kinetic studies identify intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction and shed light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.
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http://dx.doi.org/10.1016/j.celrep.2019.03.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545893PMC
April 2019

Computer design of microfluidic mixers for protein/RNA folding studies.

PLoS One 2018 20;13(6):e0198534. Epub 2018 Jun 20.

Department of Mechanical and Industrial Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States of America.

Kinetic studies of biological macromolecules increasingly use microfluidic mixers to initiate and monitor reaction progress. A motivation for using microfluidic mixers is to reduce sample consumption and decrease mixing time to microseconds. Some applications, such as small-angle x-ray scattering, also require large (>10 micron) sampling areas to ensure high signal-to-noise ratios and to minimize parasitic scattering. Chaotic to marginally turbulent mixers are well suited for these applications because this class of mixers provides a good middle ground between existing laminar and turbulent mixers. In this study, we model various chaotic to marginally turbulent mixing concepts such as flow turning, flow splitting, and vortex generation using computational fluid dynamics for optimization of mixing efficiency and observation volume. Design iterations show flow turning to be the best candidate for chaotic/marginally turbulent mixing. A qualitative experimental test is performed on the finalized design with mixing of 10 M urea and water to validate the flow turning unsteady mixing concept as a viable option for RNA and protein folding studies. A comparison of direct numerical simulations (DNS) and turbulence models suggests that the applicability of turbulence models to these flow regimes may be limited.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198534PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010218PMC
December 2018

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors.

Structure 2018 01 21;26(1):106-117.e6. Epub 2017 Dec 21.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address:

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.
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http://dx.doi.org/10.1016/j.str.2017.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752578PMC
January 2018

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY.

Sci Rep 2017 03 8;7:44116. Epub 2017 Mar 8.

Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.
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http://dx.doi.org/10.1038/srep44116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341065PMC
March 2017

Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure.

Biochemistry 2017 02 16;56(8):1085-1094. Epub 2017 Feb 16.

Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.

Fluorescence spectroscopy, relying on intrinsic protein fluorophores, is one of the most widely used methods for studying protein folding, protein-ligand interactions, and protein dynamics. Tryptophan is usually the fluorophore of choice, given its sensitivity to its environment and having the highest quantum yield of the natural amino acids; however, changes in tryptophan fluorescence can be difficult to interpret in terms of specific structural changes. The introduction of quenchers of tryptophan fluorescence can provide information about specific structures, particularly if quenching is short-range; however, the most commonly employed quencher is histidine, and it is effective only when the imidazole side chain is protonated, thus limiting the pH range over which this approach can be employed. In addition, histidine is not always a conservative substitution and is likely to be destabilizing if inserted into the hydrophobic core of proteins. Here we illustrate the use of a Trp-selenomethionine (M) pair as a specific probe of protein structure. M requires a close approach to Trp to quench its fluorescence, and this effect can be exploited to design specific probes of α-helix and β-sheet formation. The approach is illustrated using equilibrium and time-resolved fluorescence measurements of designed peptides and globular proteins. M is easily incorporated into proteins and provides a conservative replacement for hydrophobic side chains, and M quenching of Trp fluorescence is pH-independent. The oxidized form of M, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.
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http://dx.doi.org/10.1021/acs.biochem.6b01000DOI Listing
February 2017

Dye-Sensitized Core/Active Shell Upconversion Nanoparticles for Optogenetics and Bioimaging Applications.

ACS Nano 2016 Jan 11;10(1):1060-6. Epub 2016 Jan 11.

The Molecular Foundry, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.

Near-infrared (NIR) dye-sensitized upconversion nanoparticles (UCNPs) can broaden the absorption range and boost upconversion efficiency of UCNPs. Here, we achieved significantly enhanced upconversion luminescence in dye-sensitized core/active shell UCNPs via the doping of ytterbium ions (Yb(3+)) in the UCNP shell, which bridged the energy transfer from the dye to the UCNP core. As a result, we synergized the two most practical upconversion booster effectors (dye-sensitizing and core/shell enhancement) to amplify upconversion efficiency. We demonstrated two biomedical applications using these UCNPs. By using dye-sensitized core/active shell UCNP embedded poly(methyl methacrylate) polymer implantable systems, we successfully shifted the optogenetic neuron excitation window to a biocompatible and deep tissue penetrable 800 nm wavelength. Furthermore, UCNPs were water-solubilized with Pluronic F127 with high upconversion efficiency and can be imaged in a mouse model.
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http://dx.doi.org/10.1021/acsnano.5b06383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913696PMC
January 2016

Selenomethionine, p-cyanophenylalanine pairs provide a convenient, sensitive, non-perturbing fluorescent probe of local helical structure.

Chem Commun (Camb) 2016 Feb;52(10):2055-8

Department of Chemistry, Stony Brook University, Stony Brook, New York, 11794-3400, USA. and Graduate Program in Biochemistry & Structural Biology, Stony Brook University, Stony Brook, New York, 11794-3400, USA.

The use of selenomethionine (MSe)-p-cyanophenylalanine (FCN) pairs to probe protein structure is demonstrated. MSe quenches FCN fluorescence via electron transfer. Both residues can be incorporated recombinantly or by peptide synthesis. Time-resolved and steady-state fluorescence measurements demonstrate that MSe-FCN pairs provide specific local probes of helical structure.
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http://dx.doi.org/10.1039/c5cc08232cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755734PMC
February 2016

Sequential Closure of Loop Structures Forms the Folding Nucleus during the Refolding Transition of the Escherichia coli Adenylate Kinase Molecule.

Biochemistry 2016 Jan 23;55(1):79-91. Epub 2015 Dec 23.

The Goodman Faculty of Life Sciences, Bar Ilan University , Ramat Gan, Israel 52900.

The ensemble of conformers of globular protein molecules immediately following transfer from unfolding to folding conditions is assumed to be collapsed though still disordered, as the first steps of the folding pathway are initiated. In order to test the hypothesis that long loop closure transitions are part of the initiation of the folding pathway, our groups are studying the initiation of the folding transition of a model protein by time-resolved excitation energy transfer (trFRET) detected fast kinetics experiments. Site-specific double labeling is used to study the timing of conformational transitions of individual loop forming chain segments at the microsecond time regime. Previously, it was shown that at least three long loops in the Escherichia coli adenylate kinase (AK) molecule close within the first 5 ms of folding of AK, while the main global folding transition occurs in a time regime of seconds. In order to enhance the time resolution of the kinetics experiments to the microsecond time regime and determine the rate of closure of the two N terminal loops (loop I residues 1-26 and loop II residues 29-72), we applied a continuous flow based double kinetics experiment. These measurements enabled us to obtain a microsecond series of transient time dependent distributions of distances between the ends of the labeled loops. Analysis of the trFRET experiments show that the N terminal loop (loop I) is closed within less than 60 μs after the initiation of refolding. Loop II is also mostly closed within that time step but shows an additional small reduction of the mean end-to-end distance in a second phase at a rate of 0.005 μs(-1). This second phase can either reflect tightening of a loosely closed loop in the ensemble of conformers or may reflect two subpopulations in the ensemble, which differ in the rate of closure of loop II, but not in the rate of closure of loop I. This study shows the very fast closure of long loops in the otherwise disordered backbone and fine details of the very early hidden pretransition state steps that are essential for the fast and efficient folding of the protein molecule.
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http://dx.doi.org/10.1021/acs.biochem.5b00849DOI Listing
January 2016

Tailoring dye-sensitized upconversion nanoparticle excitation bands towards excitation wavelength selective imaging.

Nanoscale 2015 Nov 26;7(44):18424-8. Epub 2015 Oct 26.

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, 116012, People's Republic of China.

One of the key roadblocks in UCNP development is its extremely limited choices of excitation wavelengths. We report a generic design to program UCNPs to possess highly tunable dye characteristic excitation bands. Using such distinctive properties, we were able to develop a new excitation wavelength selective security imaging. This work unleashed the greater freedom of the excitation wavelengths of the upconversion nanoparticles and we believe it is a game-changer in the field and this method will enable numerous applications that are currently limited by existing UCNPs.
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http://dx.doi.org/10.1039/c5nr05437kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636449PMC
November 2015

Methods for analysis of size-exclusion chromatography-small-angle X-ray scattering and reconstruction of protein scattering.

J Appl Crystallogr 2015 Aug 8;48(Pt 4):1102-1113. Epub 2015 Jul 8.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA ; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC-SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC-SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD-LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known as (), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.
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http://dx.doi.org/10.1107/S1600576715010420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520288PMC
August 2015

Non-native structure appears in microseconds during the folding of E. coli RNase H.

J Mol Biol 2015 Jan 13;427(2):443-53. Epub 2014 Oct 13.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3220, USA; California Institute for Quantitative Biosciences-Berkeley, University of California, Berkeley, Berkeley, CA 94720-3220, USA; Department of Molecular and Cell Biology-Berkeley, University of California, Berkeley, Berkeley, CA 94720-3220, USA. Electronic address:

The folding pathway of Escherichia coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous-flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60 μs dead time). Two folding intermediates are observed, the second of which is the previously characterized I(core) millisecond intermediate. The new earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, I(core). Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, which complements previous hydrogen-exchange studies that monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamics simulations of protein folding.
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http://dx.doi.org/10.1016/j.jmb.2014.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624390PMC
January 2015

Modulation of frustration in folding by sequence permutation.

Proc Natl Acad Sci U S A 2014 Jul 7;111(29):10562-7. Epub 2014 Jul 7.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605;

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.
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http://dx.doi.org/10.1073/pnas.1324230111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115504PMC
July 2014

Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS.

J Mol Biol 2014 May 4;426(9):1980-94. Epub 2014 Mar 4.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59-heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.
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http://dx.doi.org/10.1016/j.jmb.2014.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010103PMC
May 2014

SAXS/SANS probe of intermolecular interactions in concentrated protein solutions.

Biophys J 2014 Feb;106(4):771-3

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts.

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http://dx.doi.org/10.1016/j.bpj.2014.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944975PMC
February 2014

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam.

J Synchrotron Radiat 2013 Nov 1;20(Pt 6):820-5. Epub 2013 Oct 1.

BioCAT, CSRRI and Department BCS, Illinois Institute of Technology, 3101 South Dearborn, Chicago, IL 60616, USA.

Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam sizes delivered by typical SAXS beamlines. These limitations can be overcome using optics to focus X-rays to the micrometer size range providing that beam divergence and photon flux suitable for performing SAXS experiments can be maintained. Such micro-SAXS in combination with microfluidic devices would be an attractive probe for time-resolved studies. Here, the development of a high-duty-cycle scanning microsecond-time-resolution SAXS capability, built around the Kirkpatrick-Baez mirror-based microbeam system at the Biophysics Collaborative Access Team (BioCAT) beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is reported. A detailed description of the microbeam small-angle-scattering instrument, the turbulent flow mixer, as well as the data acquisition and control and analysis software is provided. Results are presented where this apparatus was used to study the folding of cytochrome c. Future prospects for this technique are discussed.
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http://dx.doi.org/10.1107/S0909049513021833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795536PMC
November 2013

Advances in turbulent mixing techniques to study microsecond protein folding reactions.

Biopolymers 2013 Nov;99(11):888-96

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA, 01605.

Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps, and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10's of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer, Circular Dichroism, Small-Angle X-ray Scattering, Hydrogen Exchange followed by Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy, Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy, has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of the CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, which are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.
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http://dx.doi.org/10.1002/bip.22355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843316PMC
November 2013

Native state conformational heterogeneity of HP35 revealed by time-resolved FRET.

J Phys Chem B 2012 Sep 27;116(35):10631-8. Epub 2012 Aug 27.

Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (Phe(CN)), which are modulated by a nearby quencher (e.g., tryptophan or 7-azatryptophan) through the mechanism of fluorescence resonance energy transfer (FRET), can be used to detect protein conformational heterogeneity. This method is based on the notion that protein conformations having different donor-acceptor distances and interconverting slowly compared to the fluorescence lifetime of the donor (Phe(CN)) would exhibit different donor fluorescence lifetimes. Our results provide strong evidence suggesting that the native free energy basin of HP35 is populated with conformations that differ mostly in the position and mean helicity of the C-terminal helix. This finding is consistent with several previous experimental and computational studies. Moreover, this result holds strong implications for computational investigation of the folding mechanism of HP35.
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http://dx.doi.org/10.1021/jp211296eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438901PMC
September 2012

Microsecond subdomain folding in dihydrofolate reductase.

J Mol Biol 2011 Jul 30;410(2):329-42. Epub 2011 Apr 30.

Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.
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http://dx.doi.org/10.1016/j.jmb.2011.04.057DOI Listing
July 2011

Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering.

Biopolymers 2011 Aug 25;95(8):550-8. Epub 2011 Mar 25.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Small-angle X-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this minireview, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow-based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.
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http://dx.doi.org/10.1002/bip.21628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278217PMC
August 2011

Metal-free ALS variants of dimeric human Cu,Zn-superoxide dismutase have enhanced populations of monomeric species.

PLoS One 2010 Apr 9;5(4):e10064. Epub 2010 Apr 9.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852398PMC
April 2010

Metal deficiency increases aberrant hydrophobicity of mutant superoxide dismutases that cause amyotrophic lateral sclerosis.

J Biol Chem 2009 Oct 3;284(40):27746-58. Epub 2009 Aug 3.

Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu(2+), even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.
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http://dx.doi.org/10.1074/jbc.M109.043729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785702PMC
October 2009

The folding free-energy surface of HIV-1 protease: insights into the thermodynamic basis for resistance to inhibitors.

J Mol Biol 2009 Apr 6;387(4):1002-16. Epub 2009 Jan 6.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric beta-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR(*), was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.
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http://dx.doi.org/10.1016/j.jmb.2008.12.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2756696PMC
April 2009
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