Publications by authors named "Francesca Massi"

28 Publications

  • Page 1 of 1

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

Analysis of Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

Evol Bioinform Online 2021 5;17:11769343211014167. Epub 2021 May 5.

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

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two sets of SARS-CoV-2 genomic sequences to identify emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, 20 variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5' untranslated region (UTR), including a group of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-ss molecular switch in the 3'UTR. Finally, 5 variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M (stem loop 2 m) selfish genetic element, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. Our analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences.
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http://dx.doi.org/10.1177/11769343211014167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114311PMC
May 2021

Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

bioRxiv 2020 Jun 30. Epub 2020 Jun 30.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5'UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3'UTR. Finally, five variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.
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http://dx.doi.org/10.1101/2020.05.27.120105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302204PMC
June 2020

A Disorder-to-Order Transition Mediates RNA Binding of the Caenorhabditis elegans Protein MEX-5.

Biophys J 2020 04 19;118(8):2001-2014. Epub 2020 Mar 19.

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

CCCH-type tandem zinc finger (TZF) domains are found in many RNA-binding proteins (RBPs) that regulate the essential processes of post-transcriptional gene expression and splicing through direct protein-RNA interactions. In Caenorhabditis elegans, RBPs control the translation, stability, or localization of maternal messenger RNAs required for patterning decisions before zygotic gene activation. MEX-5 (Muscle EXcess) is a C. elegans protein that leads a cascade of RBP localization events that is essential for axis polarization and germline differentiation after fertilization. Here, we report that at room temperature, the CCCH-type TZF domain of MEX-5 contains an unstructured zinc finger that folds upon binding of its RNA target. We have characterized the structure and dynamics of the TZF domain of MEX-5 and designed a variant MEX-5 in which both fingers are fully folded in the absence of RNA. Within the thermal range experienced by C. elegans, the population of the unfolded state of the TZF domain of MEX-5 varies. We observe that the TZF domain becomes less disordered at lower temperatures and more disordered at higher temperatures. However, in the temperature range in which C. elegans is fertile, when MEX-5 needs to be functional, only one of the two zinc fingers is folded.
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http://dx.doi.org/10.1016/j.bpj.2020.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175634PMC
April 2020

Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein.

Proc Natl Acad Sci U S A 2019 04 15;116(14):6806-6811. Epub 2019 Mar 15.

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

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.
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http://dx.doi.org/10.1073/pnas.1818744116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452746PMC
April 2019

Characterization of TDP-43 RRM2 Partially Folded States and Their Significance to ALS Pathogenesis.

Biophys J 2018 11 21;115(9):1673-1680. Epub 2018 Sep 21.

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

The human protein TDP-43 is a major component of the cellular aggregates found in amyotrophic lateral sclerosis and other neurodegenerative diseases. Insoluble cytoplasmic aggregates isolated from the brain of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients contain ubiquitinated, hyperphosphorylated, and N-terminally truncated TDP-43. Truncated fragments of TDP-43 identified from patient tissues contain part of the second RNA recognition motif (RRM2) and the disordered C-terminus, indicating that both domains can be involved in aggregation and toxicity. Here, we focus on RRM2. Using all-atom replica-averaged metadynamics simulations with NMR chemical shift restraints, we characterized the atomic structure of non-native states of RRM2, sparsely populated under native conditions. These structures reveal the exposure to the solvent of aggregation-prone peptide regions, normally buried in the native state, supporting a role in aggregation for the partially folded states of RRM2.
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http://dx.doi.org/10.1016/j.bpj.2018.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224623PMC
November 2018

Characterizing Protein Dynamics with NMR R Relaxation Experiments.

Methods Mol Biol 2018 ;1688:205-221

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556, USA.

The measurement of R , the longitudinal relaxation rate constant in the rotating frame, is one of the few available methods to characterize the μs-ms functional dynamics of biomolecules. Here, we focus on N R experiments for protein NH groups. We present protocols for both on- and off-resonance N R measurements needed for relaxation dispersion studies, and describe the data analysis for extracting kinetic and thermodynamic parameters characterizing the motional processes.
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http://dx.doi.org/10.1007/978-1-4939-7386-6_10DOI Listing
July 2018

Structural Rearrangement upon Fragmentation of the Stability Core of the ALS-Linked Protein TDP-43.

Biophys J 2017 Aug;113(3):540-549

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

Amyotrophic lateral sclerosis (ALS) is the most common adult degenerative motor neuron disease. Experimental evidence indicates a direct role of transactive-response DNA-binding protein 43 (TDP-43) in the pathology of ALS and other neurodegenerative diseases. TDP-43 has been identified as a major component of cytoplasmic inclusions in patients with sporadic ALS; however, the molecular basis of the disease mechanism is not yet fully understood. Fragmentation within the second RNA recognition motif (RRM2) of TDP-43 has been observed in patient tissues and may play a role in the formation of aggregates in disease. To determine the structural and dynamical changes resulting from the truncation that could lead to aggregation and toxicity, we performed molecular dynamics simulations of the full-length RRM2 domain (the stability core of TDP-43) and of a truncated variant (where residues 189-207 are deleted to mimic a site of cleavage within RRM2 found in ALS patients). Our simulations show heterogeneous structural reorganization and decreased stability of the truncated RRM2 domain compared to the full-length domain, consistent with previous experimental results. The decreased stability and structural reorganization in the truncated RRM2 result in a higher probability of protein-protein interactions through altered electrostatic surface charges and increased accessibility of hydrophobic residues (including the nuclear export sequence), providing a rationale for the increased cytoplasmic aggregation of RRM2 fragments seen in sporadic ALS patients.
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http://dx.doi.org/10.1016/j.bpj.2017.06.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550305PMC
August 2017

Structural Basis of the Disorder in the Tandem Zinc Finger Domain of the RNA-Binding Protein Tristetraprolin.

J Chem Theory Comput 2016 Oct 9;12(10):4717-4725. Epub 2016 Sep 9.

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

Tristetraprolin (TTP) and TIS11d are two human RNA-binding proteins that belong to the CCCH-type tandem zinc finger family. In the RNA-free state, TIS11d coordinates a zinc ion in each of its two fingers, while TTP coordinates a single zinc ion with the N-terminal zinc finger. We have previously identified three residues, located in the C-terminal half of a short α-helix in the second zinc finger, that control how structured the RNA-binding domain is in these two proteins: Y151, L152, and Q153 in TTP and H201, T202, and I203 in TIS11d. Here, we have used molecular dynamics, NMR spectroscopy, and other biochemical methods to investigate the role of these three residues in the stability of the RNA-binding domain. We found that the intrahelical hydrogen bond formed by the T202 hydroxyl group in the C-terminal zinc finger of TIS11d is necessary to allow for π-π stacking between the side chains of a conserved phenylalanine and the zinc-coordinating histidine. We demonstrated that the lack of this hydrogen bond in TTP is responsible for the reduced zinc affinity of the C-terminal zinc finger.
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http://dx.doi.org/10.1021/acs.jctc.6b00150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137377PMC
October 2016

Three Residues Make an Evolutionary Switch for Folding and RNA-Destabilizing Activity in the TTP Family of Proteins.

ACS Chem Biol 2016 Feb 14;11(2):435-43. Epub 2015 Dec 14.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School , 364 Plantation Street, Worcester, Massachusetts 01605, United States.

Tristetraprolin (TTP) binds to mRNA transcripts to promote their degradation. The TTP protein family in humans includes two other proteins, TIS11b and TIS11d. All three proteins contain a highly homologous RNA binding domain (RBD) that consists of two CCCH zinc fingers (ZFs). Both ZFs are folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold. The focus of this study is to understand the origin and biological significance of the structural differences of the RBD. We identified three residues that affect the affinity for the structural Zn(2+) and determine the folding of ZF2 in the absence of RNA. We observed that the mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured RBD of TIS11d, indicating that differences in the folded state of the RBD affect the activity of the proteins in the cell.
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http://dx.doi.org/10.1021/acschembio.5b00639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129185PMC
February 2016

Probing the structural and dynamical effects of the charged residues of the TZF domain of TIS11d.

Biophys J 2015 Mar;108(6):1503-1515

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

A member of the TTP family of proteins, TIS11d binds RNA with high specificity using a pair of CCCH-type tandem zinc fingers separated by a 18 residue long linker. Our previous work showed that the formation of hydrogen bonds between the C-terminal residue E220 and the residues of the linker region stabilized a compact structure of TIS11d in the absence of RNA. To investigate the role of the C-terminal residues in the structure of unbound TIS11d, the E220A mutant and the truncation mutant lacking the last two residues (D219/E220) were studied using molecular dynamics, NMR spectroscopy, and biochemical methods. This study confirmed the importance of the charged residues D219 and E220 in maintaining structural stability in unbound TIS11d and elucidated the underlying physical mechanisms. We observed a greater structural heterogeneity for the residues of the linker in the molecular dynamics trajectories of both mutant proteins relative to the wild-type. This heterogeneity was more pronounced in the D219/E220 deletion mutant than in the E220A mutant, indicating that a greater reduction of the charge of the C-terminus results in greater flexibility. In agreement with the increased flexibility and the reduced number of negatively charged residues of the D219/E220 deletion mutant, we measured more unfavorable entropic and a more favorable enthalpic contribution to the free energy of RNA binding in the mutant than in the wild-type protein. The relative orientation of the zinc fingers was stabilized by the electrostatic interaction between E220 and positively charged residues of the linker in TIS11d. In the E220A mutant, the relative orientation of the zinc fingers was less constrained, whereas in the D219/E220 deletion mutant, little orientational preference was observed. We posit that favorable electrostatic interactions provide a mechanism to promote preferential orientation of separate domains without imposing structural rigidity.
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http://dx.doi.org/10.1016/j.bpj.2015.01.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375430PMC
March 2015

A conserved three-nucleotide core motif defines Musashi RNA binding specificity.

J Biol Chem 2014 Dec 3;289(51):35530-41. Epub 2014 Nov 3.

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

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins.
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http://dx.doi.org/10.1074/jbc.M114.597112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271237PMC
December 2014

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Biochemistry 2014 Nov 14;53(46):7199-210. Epub 2014 Nov 14.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts , Worcester, Massachusetts 01605, United States.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.
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http://dx.doi.org/10.1021/bi500591sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245988PMC
November 2014

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite.

Elife 2014 Jun 16;3. Epub 2014 Jun 16.

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

Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18-22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state.
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http://dx.doi.org/10.7554/eLife.02848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094780PMC
June 2014

Clusters of branched aliphatic side chains serve as cores of stability in the native state of the HisF TIM barrel protein.

J Mol Biol 2013 Mar 16;425(6):1065-81. Epub 2013 Jan 16.

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

Imidazole-3-glycerol phosphate synthase is a heterodimeric allosteric enzyme that catalyzes consecutive reactions in imidazole biosynthesis through its HisF and HisH subunits. The unusually slow unfolding reaction of the isolated HisF TIM barrel domain from the thermophilic bacteria, Thermotoga maritima, enabled an NMR-based site-specific analysis of the main-chain hydrogen bonds that stabilize its native conformation. Very strong protection against exchange with solvent deuterium in the native state was found in a subset of buried positions in α-helices and pervasively in the underlying β-strands associated with a pair of large clusters of isoleucine, leucine and valine (ILV) side chains located in the α7(βα)8(βα)1-2 and α2(βα)3-6β7 segments of the (βα)8 barrel. The most densely packed region of the large cluster, α3(βα)4-6β7, correlates closely with the core of stability previously observed in computational, protein engineering and NMR dynamics studies, demonstrating a key role for this cluster in determining the thermodynamic and structural properties of the native state of HisF. When considered with the results of previous studies where ILV clusters were found to stabilize the hydrogen-bonded networks in folding intermediates for other TIM barrel proteins, it appears that clusters of branched aliphatic side chains can serve as cores of stability across the entire folding reaction coordinate of one of the most common motifs in biology.
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http://dx.doi.org/10.1016/j.jmb.2013.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696590PMC
March 2013

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease.

J Virol 2011 Jul 20;85(13):6106-16. Epub 2011 Apr 20.

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

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.
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http://dx.doi.org/10.1128/JVI.00377-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126519PMC
July 2011

Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα.

J Mol Biol 2011 May 17;408(5):949-70. Epub 2011 Mar 17.

Department of Biochemistry and Biophysics, Texas A&M University, 300 Olsen Boulevard, College Station, TX 77843, USA.

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a >100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.
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http://dx.doi.org/10.1016/j.jmb.2011.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489171PMC
May 2011

Insights into the structural basis of RNA recognition by STAR domain proteins.

Adv Exp Med Biol 2010 ;693:37-53

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, LRB-906, Worcester, Massachusetts 01605, USA.

STAR proteins regulate diverse cellular processes and control numerous developmental events. They function at the post-transcriptional level by regulating the stability, sub-cellular distribution, alternative splicing, or translational efficiency of specific mRNA targets. Significant effort has been expended to define the determinants of RNA recognition by STAR proteins, in hopes of identifying new mRNA targets that contribute their role in cellular metabolism and development. This work has lead to the extensive biochemical characterization ofthe nucleotide sequence specificity of a handful of STAR proteins. In contrast, little structural information is available to analyze the molecular basis of sequence specific RNA recognition by this protein family. This chapter reviews the relevant literature on STAR domain protein structure and provides insights into how these proteins discriminate between different RNA sequences.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034770PMC
http://dx.doi.org/10.1007/978-1-4419-7005-3_3DOI Listing
February 2011

Accurate Estimates of Free Energy Changes in Charge Mutations.

J Chem Theory Comput 2010 Jun;6(6):1884-93

Department of Physics, Clark University, 950 Main Street, Worcester, Massachusetts 01610 and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts, 55 Lake Avenue North, Worcester, Massachusetts 01655.

The ability to determine the effect of charge changes on the free energy is necessary for fundamental studies of the electrostatic contribution to protein binding and stability. Currently, calculations of differences in free energy for charge mutations carried out in systems with periodic boundary conditions must include an approximate self-energy correction that can be on the same order of magnitude as the calculated free energy change. Here, a new method for accurately calculating the free energy change associated with any alchemical mutation, regardless of charge, is presented. In this method, paired mutations of opposite charge exactly cancel the self-energy term because of its quadratic charge dependence. Since the self-energy term implicitly cancels within the method, a correction never needs to be applied, and the statistical uncertainty associated is thereby removed. An implementation procedure is described and applied to the free energy of ionic hydration and a charged amino acid mutation.
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http://dx.doi.org/10.1021/ct900565eDOI Listing
June 2010

A computational study of RNA binding and specificity in the tandem zinc finger domain of TIS11d.

Protein Sci 2010 Jun;19(6):1222-34

Department of Physics, Clark University, Worcester, Massachusetts 01610, USA.

TIS11d is a member of the CCCH-type family of tandem zinc finger (TZF) proteins; the TZF domain of TIS11d (residues 151-220) is sufficient to bind and destabilize its target mRNAs with high specificity. In this study, the TZF domain of TIS11d is simulated in an aqueous environment in both the free and RNA-bound states. Multiple nanosecond timescale molecular dynamics trajectories of TIS11d wild-type and E157R/E195K mutant with different RNA sequences were performed to investigate the molecular basis for RNA binding specificities of this TZF domain. A variety of measures of the protein structure, fluctuations, and dynamics were used to analyze the trajectories. The results of this study support the following conclusions: (1) the structure of the two fingers is maintained in the free state but a global reorientation occurs to yield a more compact structure; (2) mutation of the glutamate residues at positions 157 and 195 to arginine and lysine, respectively, affects the RNA recognition by this TIS11d mutant in agreement with the findings of Pagano et al. (J Biol Chem 2007; 282:8883-8894); and (3) we predict that the E157R/E195K mutant will present a more relaxed RNA binding specificity relative to wild-type TIS11d based on the more favorable nonsequence-specific Coulomb interaction of the two positively charged residues at positions 157 and 195 with the RNA backbone, which compensates for a partial loss of the stacking interaction of aromatic side chains with the RNA bases.
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http://dx.doi.org/10.1002/pro.401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895246PMC
June 2010

Joint composite-rotation adiabatic-sweep isotope filtration.

J Biomol NMR 2007 May 13;38(1):11-22. Epub 2007 Mar 13.

Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, NY 10032, USA.

Joint composite-rotation adiabatic-sweep isotope filters are derived by combining the composite-rotation [Stuart AC et al. (1999) J Am Chem Soc 121: 5346-5347] and adiabatic-sweep [Zwahlen C et al. (1997) J Am Chem Soc 119:6711-6721; Kupce E, Freeman R (1997) J Magn Reson 127:36-48] approaches. The joint isotope filters have improved broadband filtration performance, even for extreme values of the one-bond (1)H-(13)C scalar coupling constants in proteins and RNA molecules. An average Hamiltonian analysis is used to describe evolution of the heteronuclear scalar coupling interaction during the adiabatic sweeps within the isotope filter sequences. The new isotope filter elements permit improved selective detection of NMR resonance signals originating from (1)H spins attached to an unlabeled natural abundance component of a complex in which the other components are labeled with (13)C and (15)N isotopes.
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http://dx.doi.org/10.1007/s10858-006-9131-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2290856PMC
May 2007

Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase.

Biochemistry 2006 Sep;45(36):10787-94

Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA.

Solution NMR spin relaxation experiments and classical MD simulations are used to study the dynamics of triosephosphate isomerase (TIM) in complex with glycerol 3-phosphate (G3P). Three regions in TIM exhibit conformational transitions on the micros-ms time scale as detected by chemical exchange broadening effects in NMR spectroscopy: residue Lys 84 on helix C, located at the dimeric interface; active site loop 6; and helix G. The results indicate that the conformational exchange process affecting the residues of loop 6 is the correlated opening and closing of the loop. Distinct processes are responsible for the chemical exchange linebroadening observed in the other regions of TIM. MD simulations confirm that motions of individual residues within the active site loop are correlated and suggest that the chemical exchange processes observed for residues in helix G arise from transitions between 3(10)- and alpha-helical structures. The results of the joint NMR and MD study provide global insight into the role of conformational dynamic processes in the function of TIM.
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http://dx.doi.org/10.1021/bi060764cDOI Listing
September 2006

Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy.

Chem Rev 2006 May;106(5):1700-19

Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA.

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http://dx.doi.org/10.1021/cr0404287DOI Listing
May 2006

Temperature dependence of NMR order parameters and protein dynamics.

J Am Chem Soc 2003 Sep;125(37):11158-9

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.

The helical subdomain, HP36, of the F-actin-binding headpiece domain of chicken villin, is the smallest naturally occurring polypeptide that folds to a thermostable compact structure. Unconstrained molecular dynamics simulations and constrained molecular dynamics simulations using umbrella sampling are used to study the temperature dependence of internal motions of the backbone amide moieties of HP36. The potential of mean force (PMF) for the N-H bond vector, determined from the constrained simulations, is found to be temperature dependent. A simple analytical expression is derived that describes the temperature dependence of the PMF. The parameters of this model are obtained from the PMF, from the unconstrained molecular dynamics simulations, or from experimental values of the generalized order parameter. The results provide a linkage between experimental and theoretical measures of the temperature dependence of protein motions.
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http://dx.doi.org/10.1021/ja035605kDOI Listing
September 2003

Microsecond timescale backbone conformational dynamics in ubiquitin studied with NMR R1rho relaxation experiments.

Protein Sci 2005 Mar;14(3):735-42

Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, NY 10032, USA.

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on- and off-resonance rotating-frame 15N R1rho relaxation experiments to have a kinetic exchange rate constant of 25,000 sec(-1) at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N-cap hydrogen bonds of the alpha-helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1H-15N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin-dependent protein degradation pathway.
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http://dx.doi.org/10.1110/ps.041139505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279275PMC
March 2005

NMR R1 rho rotating-frame relaxation with weak radio frequency fields.

J Am Chem Soc 2004 Feb;126(7):2247-56

Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA.

NMR spin relaxation in the rotating frame (R(1 rho)) is one of few methods available to characterize chemical exchange kinetic processes occurring on micros-ms time scales. R(1 rho) measurements for heteronuclei in biological macromolecules generally require decoupling of (1)H scalar coupling interactions and suppression of cross-relaxation processes. Korzhnev and co-workers demonstrated that applying conventional (1)H decoupling schemes while the heteronuclei are spin-locked by a radio frequency (rf) field results in imperfect decoupling [Korzhnev, Skrynnikov, Millet, Torchia, Kay. J. Am. Chem. Soc. 2002, 124, 10743-10753]. Experimental NMR pulse sequences were presented that provide accurate measurements of R(1 rho) rate constants for radio frequency field strengths > 1000 Hz. This paper presents new two-dimensional NMR experiments that allow the use of weak rf fields, between 150 and 1000 Hz, in R(1 rho) experiments. Fourier decomposition and average Hamiltonian theory are employed to analyze the spin-lock sequence and provide a guide for the development of improved experiments. The new pulse sequences are validated using ubiquitin and basic pancreatic trypsin inhibitor (BPTI). The use of weak spin-lock fields in R(1 rho) experiments allows the study of the chemical exchange process on a wider range of time scales, bridging the gap that currently exists between Carr-Purcell-Meiboom-Gill and conventional R(1 rho) experiments. The new experiments also extend the capability of the R(1 rho) technique to study exchange processes outside the fast exchange limit.
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http://dx.doi.org/10.1021/ja038721wDOI Listing
February 2004

Structural and dynamical analysis of the hydration of the Alzheimer's beta-amyloid peptide.

J Comput Chem 2003 Jan;24(2):143-53

Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.

An analysis of the water molecules in the first solvation shell obtained from the molecular dynamics simulation of the amyloid beta(10-35)NH2 peptide and the amyloid beta(10-35)NH2E22Q "Dutch" mutant peptide is presented. The structure, energetics, and dynamics of water in the hydration shell have been investigated using a variety of measures, including the hydrogen bond network, the water residence times for all the peptide residues, the diffusion constant, experimentally determined HN amide proton exchange, and the transition probabilities for water to move from one residue to another or into the bulk. The results of the study indicate that: (1) the water molecules at the peptide-solvent interface are organized in an ordered structure similar for the two peptide systems but different from that of the bulk, (2) the peptide structure inhibits diffusion perpendicular to the peptide surface by a factor of 3 to 5 relative to diffusion parallel to the peptide surface, which is comparable to diffusion of bulk water, (3) water in the first solvation shell shows dynamical relaxation on fast (1-2 ps) and slow (10-40 ps) time scales, (4) a novel solvent relaxation master equation is shown to capture the details of the fast relaxation of water in the peptide's first solvation shell, (5) the interaction between the peptide and the solvent is stronger in the wild type than in the E22Q mutant peptide, in agreement with earlier results obtained from computer simulations [Massi, F.; Straub, J. E. Biophys J 2001, 81, 697] correlated with the observed enhanced activity of the E22Q mutant peptide.
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http://dx.doi.org/10.1002/jcc.10101DOI Listing
January 2003

Charge states rather than propensity for beta-structure determine enhanced fibrillogenesis in wild-type Alzheimer's beta-amyloid peptide compared to E22Q Dutch mutant.

Protein Sci 2002 Jul;11(7):1639-47

Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA 02215, USA.

The activity of the Alzheimer's amyloid beta-peptide is a sensitive function of the peptide's sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimer's amyloid beta-peptide relative to that of the wild-type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of beta structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Abeta(10-35)-peptide in aqueous solution. The simulation results indicate that the propensity for formation of beta-structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of "flickering" of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of beta-structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373666PMC
http://dx.doi.org/10.1110/ps.3150102DOI Listing
July 2002
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