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REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022). 1 ., Competing Interests: Declaration of interests J.D.B. is a cofounder and holds equity shares in SiteOne Therapeutics, Inc., a start-up company interested in developing subtype-selective modulators of sodium channels. Patent applications have been filed for this work., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20-30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a "budding power" of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20-30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdan's generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Heat stress triggers a specific set of proteins in budding yeast to form solid-like biomolecular condensates, which are dispersed by molecular chaperones. Here, we describe a protocol to study the kinetics of chaperone-facilitated condensate dispersal using biochemical reconstitution and fluorescence anisotropy. Although the current protocol is tailored to study heat-induced condensates of poly(A)-binding protein (Pab1), the protocol can be modified to study any protein which shows differential substrate binding activity upon condensation. For complete details on the use and execution of this protocol, please refer to Yoo et al. (2022)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

BAX activation techniques are crucial to studying the intrinsic pathway of apoptosis- thousands of pro-apoptotic signals converge on BAX activation. Current methodologies are predominantly limited to membrane permeabilization studies, which assess endpoint functionality of oligomeric BAX, but overlook early activation steps of cytosolic BAX. Here we detail FLAMBE: a fluorescence polarization ligand assay for monitoring BAX early-activation in solution. We also describe a dual-metric parameterization strategy for distillation of kinetic data and comparative analyses when studying candidate ligands. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The repurposing of structurally conserved protein domains in different functional contexts is thought to be a driving force in the evolution of complex protein interaction networks. The BTB/POZ domain is such a versatile binding module that occurs over 200 times in the human proteome with diverse protein-specific adaptations. In BTB-zinc-finger transcription factors, the BTB domain drives homo- and heterodimerization as well as interactions with non-BTB-domain-containing proteins. Which mechanisms encode specificity in these interactions at a structural level is incompletely understood. Here, we uncover an atypical peptide-binding site in the BTB domain of the MYC-interacting zinc-finger protein 1 (MIZ1) that arises from local flexibility of the core BTB fold and may provide a target site for MIZ1-directed therapeutic approaches. Intriguingly, the identified binding mode requires the BTB domain to be in a homodimeric state, thus holding opportunities for functional discrimination between homo- and heterodimers of MIZ1 in the cell., Competing Interests: Declaration of interests S.L. is a member of the advisory board of Structure., (Copyright © 2021. Published by Elsevier Ltd.)

Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment. • A fluorescence polarization-based assay to monitor E3 ligase activity was developed • Small molecules that activate E6AP/UBE3A were identified in a high-throughput screen • The compounds rescue catalytically impaired Angelman syndrome-derived E6AP variants • The compounds induce conformational rearrangements of E6AP as revealed by XL-MS Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Here, several compounds are identified that rescue the E3 activity of certain AS-derived E6AP variants. These compounds represent potential lead structures for the design of drugs for AS treatment. [ABSTRACT FROM AUTHOR]

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na 2 SO 4 ) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The core subunit of the MORF acetyltransferase complex BRPF1 contains a unique combination of zinc fingers, including a plant homeodomain (PHD) finger followed by a zinc knuckle and another PHD finger, which together form a PZP domain (BRPF1 PZP ). BRPF1 PZP has been shown to bind to the nucleosome and make contacts with both histone H3 tail and DNA. Here, we describe biophysical and structural methods for characterization of the interactions between BRPF1 PZP , H3 tail, DNA, and the intact nucleosome. For complete details on the use and execution of this protocol, please refer to Klein et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (〈E FRET exp 〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈E FRET MD 〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ 2 〉 = 0.17 ± 0.16 and 〈|κ|〉 = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Förster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (k FRET ), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (r ET ) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Membrane viscosity and hydration levels characterize the biophysical properties of biological membranes and are reflected in the rate and extent of solvent relaxation, respectively, of environmentally sensitive fluorophores such as Laurdan. Here, we first developed a method for a time-resolved general polarization (GP) analysis with fluorescence-lifetime imaging microscopy that captures both the extent and rate of Laurdan solvent relaxation. We then conducted time-resolved GP measurements with Laurdan-stained model membranes and cell membranes. These measurements revealed that cholesterol levels in lipid vesicles altered membrane hydration and viscosity, whereas curvature had little effect on either parameter. We also applied the method to the plasma membrane of live cells using a supercritical angle fluorescence objective, to our knowledge the first time fluorescence-lifetime imaging microscopy images were generated with supercritical angle fluorescence. Here, we found that local variations in membrane cholesterol most likely account for the heterogeneity of Laurdan lifetime in plasma membrane. In conclusion, time-resolved GP measurements provide additional insights into the biophysical properties of membranes., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

LIN28 is an RNA-binding protein that regulates the maturation of the let-7 family of microRNAs by bipartite interactions with let-7 precursors through its two distinct cold shock and zinc-knuckle domains. Through inhibition of let-7 biogenesis, LIN28 functions as a pluripotency factor, as well as a driver of tumorigenesis. Here, we report a fluorescence polarization assay to identify small-molecule inhibitors for both domains of LIN28 involved in let-7 interactions. Of 101,017 compounds screened, six inhibit LIN28:let-7 binding and impair LIN28-mediated let-7 oligouridylation. Upon further characterization, we demonstrate that the LIN28 inhibitor TPEN destabilizes the zinc-knuckle domain of LIN28, while LI71 binds the cold shock domain to suppress LIN28's activity against let-7 in leukemia cells and embryonic stem cells. Our results demonstrate selective pharmacologic inhibition of individual domains of LIN28 and provide a foundation for therapeutic inhibition of the let-7 biogenesis pathway in LIN28-driven diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Topoisomerase IIβ binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between an FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1; however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest that similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Spatiotemporal control of the cAMP signaling pathway is governed by both hormonal stimulation of cAMP generation by adenylyl cyclases (activation phase) and cAMP hydrolysis by phosphodiesterases (PDEs) (termination phase). The termination phase is initiated by PDEs actively targeting the protein kinase A (PKA) R-subunit through formation of a PDE-PKAR-cyclic adenosine monophosphate (cAMP) complex (the termination complex). Our results using PDE8 as a model PDE, reveal that PDEs mediate active hydrolysis of cAMP bound to its receptor RIα by enhancing the enzymatic activity. This accelerated cAMP turnover occurs via formation of a stable PDE8-RIα complex, where the protein-protein interface forms peripheral contacts and the central ligand cements this ternary interaction. The basis for enhanced catalysis is active translocation of cAMP from its binding site on RIα to the hydrolysis site on PDE8 through direct "channeling." Our results reveal cAMP channeling in the PDE8-RIα complex and a molecular description of how this channel facilitates processive hydrolysis of unbound cAMP. Thus, unbound cAMP maintains the PDE8-RIα complex while being hydrolyzed, revealing an undiscovered mode for amplification of PKA activity by cAMP-mediated sequestration of the R-subunit by PDEs. This novel regulatory mode explains the paradox of cAMP signal amplification by accelerated PDE-mediated cAMP turnover. This highlights how target effector proteins of small-molecule ligands can promote enzyme-mediated ligand hydrolysis by scaffolding effects. Enhanced activity of the PDE8-RIα complex facilitates robust desensitization, allowing the cell to respond to dynamic levels of cAMP rather than steady-state levels. The PDE8-RIα complex represents a new class of PDE-based complexes for specific drug discovery targeting the cAMP signaling pathway., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based processes. End-binding (EB) proteins are considered master regulators of +TIP interaction networks, since they autonomously track growing microtubule ends and recruit a plethora of proteins to this location. Two major EB-interacting elements have been described: CAP-Gly domains and linear SxIP sequence motifs. Here, we identified LxxPTPh as a third EB-binding motif that enables major +TIPs to interact with EBs at microtubule ends. In contrast to EB-SxIP and EB-CAP-Gly, the EB-LxxPTPh binding mode does not depend on the C-terminal tail region of EB. Our study reveals that +TIPs developed additional strategies besides CAP-Gly and SxIP to target EBs at growing microtubule ends. They further provide a unique basis to discover novel +TIPs, and to dissect the role of key interaction nodes and their differential regulation for hierarchical +TIP network organization and function in eukaryotic organisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Myosin VI is critical for cargo trafficking and sorting during early endocytosis and autophagosome maturation, and abnormalities in these processes are linked to cancers, neurodegeneration, deafness, and hypertropic cardiomyopathy. We identify a structured domain in myosin VI, myosin VI ubiquitin-binding domain (MyUb), that binds to ubiquitin chains, especially those linked via K63, K11, and K29. Herein, we solve the solution structure of MyUb and MyUb:K63-linked diubiquitin. MyUb folds as a compact helix-turn-helix-like motif and nestles between the ubiquitins of K63-linked diubiquitin, interacting with distinct surfaces of each. A nine-amino-acid extension at the C-terminal helix (Helix2) of MyUb is required for myosin VI interaction with endocytic and autophagic adaptors. Structure-guided mutations revealed that a functional MyUb is necessary for optineurin interaction. In addition, we found that an isoform-specific helix restricts MyUb binding to ubiquitin chains. This work provides fundamental insights into myosin VI interaction with ubiquitinated cargo and functional adaptors., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The bioactive form of vitamin D [1,25(OH)2D3] regulates mineral and bone homeostasis and exerts potent anti-inflammatory and antiproliferative properties through binding to the vitamin D receptor (VDR). The 3D structures of the VDR ligand-binding domain with 1,25(OH)2D3 or gemini analogs unveiled the molecular mechanism underlying ligand recognition. On the basis of structure-function correlations, we generated a point-mutated VDR (VDR(gem)) that is unresponsive to 1,25(OH)2D3, but the activity of which is efficiently induced by the gemini ligands. Moreover, we show that many VDR target genes are repressed by unliganded VDR(gem) and that mineral ion and bone homeostasis are more impaired in VDR(gem) mice than in VDR null mice, demonstrating that mutations abolishing VDR ligand binding result in more severe skeletal defects than VDR null mutations. As gemini ligands induce VDR(gem) transcriptional activity in mice and normalize their serum calcium levels, VDR(gem) is a powerful tool to further unravel both liganded and unliganded VDR signaling., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The precise determination of the position of fluorescent labels is essential for the quantitative study of biomolecular structures by various localization microscopy techniques. Localization by stepwise photobleaching is especially suited for measuring nanometer-scale distances between two labels; however, the precision of this method has remained elusive. Here, we show that shot noise from other emitters and error propagation compromise the localization precision in stepwise photobleaching. Incorporation of point spread function-shaped shot noise into the variance term in the Fisher matrix yielded fundamental Cràmer-Rao lower bounds (CRLBs) that were in general anisotropic and depended on emitter intensity and position. We performed simulations to benchmark the extent to which different analysis procedures reached these ideal CRLBs. The accumulation of noise from several images accounted for the worse localization precision in image subtraction. Propagation of fitting errors compromised the CRLBs in sequential fitting using fixed parameters. Global fitting of all images was also governed by error propagation, but made optimal use of the available information. The precision of individual distance measurements depended critically on the exact bleaching kinetics and was correctly quantified by the CRLBs. The methods presented here provide a consistent framework for quantitatively analyzing stepwise photobleaching experiments and shed light on the localization precision in some other bleaching- or blinking-assisted techniques.

Multidomain scaffold proteins serve as hubs in the signal transduction network. By physically colocalizing sequential steps in a transduction pathway, scaffolds catalyze and direct incoming signals. Much is known about binary interactions with individual domains, but it is unknown whether "scaffolding activity" is predictable from pairwise affinities. Here, we characterized multivalent binding to PSD-95, a scaffold protein containing three PDZ domains connected in series by disordered linkers. We used single molecule fluorescence to watch soluble PSD-95 recruit diffusing proteins to a surface-attached receptor cytoplasmic domain. Different ternary complexes showed unique concentration dependence for scaffolding despite similar pairwise affinity. The concentration dependence of scaffolding activity was not predictable based on binary interactions. PSD-95 did not stabilize specific complexes, but rather increased the frequency of transient binding events. Our results suggest that PSD-95 maintains a loosely connected pleomorphic ensemble rather than forming a stereospecific complex containing all components., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The behavior of fluorescence anisotropy and polarization in systems with multiple dyes is well known. Homo-FRET and its consequent energy migration cause the fluorescence anisotropy to decrease as the number of like fluorophores within energy transfer distance increases. This behavior is well understood when all subunits within a cluster are saturated with fluorophores. However, incomplete labeling as might occur from a mixture of endogenous and labeled monomer units, incomplete saturation of binding sites, or photobleaching produces stochastic mixtures. Models in widespread and longstanding use that describe these mixtures apply an assumption of equal fluorescence efficiency for all sites first stated by Weber and Daniel in 1966. The assumption states that fluorophores have the same brightness when free in solution as they do in close proximity to each other in a cluster. The assumption simplifies descriptions of anisotropy trends as the fractional labeling of the cluster changes. However, fluorophores in close proximity often exhibit nonadditivity due to such things as self-quenching behavior or exciplex formation. Therefore, the anisotropy of stochastic mixtures of fluorophore clusters of a particular size will depend on the behavior of those fluorophores in clusters. We present analytical expressions for fractionally labeled clusters exhibiting a range of behaviors, and experimental results from two systems: an assembled tetrameric cluster of fluorescent proteins and stochastically labeled bovine serum albumin containing up to 24 fluorophores. The experimental results indicate that clustered species do not follow the assumption of equal fluorescence efficiency in the systems studied with clustered fluorophores showing reduced fluorescence intensity. Application of the assumption of equal fluorescence efficiency will underpredict anisotropy and consequently underestimate cluster size in these two cases. The theoretical results indicate that careful selection of the fractional labeling in strongly quenched systems will enhance opportunities to determine cluster sizes, making accessible larger clusters than are currently considered possible., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Total internal reflection fluorescence microscopy (TIRFM) is becoming an increasingly common methodology to narrow the illumination excitation thickness to study cellular process such as exocytosis, endocytosis, and membrane dynamics. It is also frequently used as a method to improve signal/noise in other techniques such as in vitro single-molecule imaging, stochastic optical reconstruction microscopy/photoactivated localization microscopy imaging, and fluorescence resonance energy transfer imaging. The unique illumination geometry of TIRFM also enables a distinct method to create an excitation field for selectively exciting fluorophores that are aligned either parallel or perpendicular to the optical axis. This selectivity has been used to study orientation of cell membranes and cellular proteins. Unfortunately, the coherent nature of laser light, the typical excitation source in TIRFM, often creates spatial interference fringes across the illuminated area. These fringes are particularly problematic when imaging large cellular areas or when accurate quantification is necessary. Methods have been developed to minimize these fringes by modulating the TIRFM field during a frame capture period; however, these approaches eliminate the possibility to simultaneously excite with a specific polarization. A new, to our knowledge, technique is presented, which compensates for spatial fringes while simultaneously permitting rapid image acquisition of both parallel and perpendicular excitation directions in ~25 ms. In addition, a back reflection detection scheme was developed that enables quick and accurate alignment of the excitation laser. The detector also facilitates focus drift compensation, a common problem in TIRFM due to the narrow excitation depth, particularly when imaging over long time courses or when using a perfusion flow chamber. The capabilities of this instrument were demonstrated by imaging membrane orientation using DiO on live cells and on lipid bilayers that were supported on a glass slide (supported lipid bilayer). The use of the approach to biological problems was illustrated by examining the temporal and spatial dynamics of exocytic vesicles., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The mitotic regulator Pin1 plays an important role in protein quality control and age-related medical conditions such as Alzheimer disease and Parkinson disease. Although its cellular role has been thoroughly investigated during the past decade, the molecular mechanisms underlying its function remain elusive. We provide evidence for interactions between the two domains of Pin1. Several residues displayed unequivocal peak splits in nuclear magnetic resonance spectra, indicative of two different conformational states in equilibrium. Pareto analysis of paramagnetic relaxation enhancement data demonstrates that the two domains approach each other upon addition of a nonpeptidic ligand. Titration experiments with phosphorylated peptides monitored by fluorescence anisotropy and chemical shift perturbation indicate that domain interactions increase Pin1's affinity toward peptide ligands. We propose this interplay of the domains and ligands to be a general mechanism for a large class of two-domain proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Nuclear hormone receptors (NRs) are major targets for pharmaceutical development. Many experiments demonstrate that their C-terminal Helix (H12) is more flexible in the ligand-binding domains (LBDs) without ligand, this increased mobility being correlated with transcription repression and human diseases. Crystal structures have been obtained in which the H12 is extended, suggesting the possibility of large amplitude H12 motions in solution. However, these structures were interpreted as possible crystallographic artifacts, and thus the microscopic nature of H12 movements is not well known. To bridge the gap between experiments and molecular models and provide a definitive picture of H12 motions in solution, extensive molecular dynamics simulations of the peroxisome proliferator-activated receptor-γ LBD, in which the H12 was bound to a fluorescent probe, were performed. A direct comparison of the modeled anisotropy decays to time-resolved fluorescence anisotropy experiments was obtained. It is shown that the decay rates are dependent on the interactions of the probe with the surface of the protein, and display little correlation with the flexibility of the H12. Nevertheless, for the probe to interact with the surface of the LBD, the H12 must be folded over the body of the LBD. Therefore, the molecular mobility of the H12 should preserve the globularity of the LBD, so that ligand binding and dissociation occur by diffusion through the surface of a compact receptor. These results advance the comprehension of both ligand-bound and ligand-free receptor structures in solution, and also guide the interpretation of time-resolved anisotropy decays from a molecular perspective, particularly by the use of simulations., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mixed lineage leukemia (MLL) fusion proteins cause oncogenic transformation of hematopoietic cells by constitutive recruitment of elongation factors to HOX promoters, resulting in overexpression of target genes. The structural basis of transactivation by MLL fusion partners remains undetermined. We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding. We determined the structure of the AF9 AHD in complex with the elongation factor AF4 and show that aliphatic residues, which are conserved in each of the AF9 binding partners, form an integral part of the hydrophobic core of the complex. Nuclear magnetic resonance relaxation measurements show that AF9 retains significant dynamic behavior which may facilitate exchange between disordered partners. We propose that AF9 functions as a signaling hub that regulates transcription through dynamic recruitment of cofactors in normal hematopoiesis and in acute leukemia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mapping the landscape of a protein's conformational space is essential to understanding its functions and regulation. The limitations of many structural methods have made this process challenging for most proteins. Here, we report that transition metal ion FRET (tmFRET) can be used in a rapid, highly parallel screen, to determine distances from multiple locations within a protein at extremely low concentrations. The distances generated through this screen for the protein maltose binding protein (MBP) match distances from the crystal structure to within a few angstroms. Furthermore, energy transfer accurately detects structural changes during ligand binding. Finally, fluorescence-derived distances can be used to guide molecular simulations to find low energy states. Our results open the door to rapid, accurate mapping and prediction of protein structures at low concentrations, in large complex systems, and in living cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Hyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entry path. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

For cells to adapt to different tissues and changes in tissue mechanics, they must be able to respond to mechanical cues by changing their gene expression patterns. Biochemical signaling pathways for these responses have been elucidated, and recent evidence points to the involvement of force-induced deformation of the nucleus. However, it is still unclear how physical cues received at the plasma membrane (PM) spatiotemporally integrate to the functional chromatin organization of the cell nucleus. To investigate this issue, we applied mechanical forces through magnetic particles adhered to the PM of single cells and mapped the accompanying changes in actin polymerization, nuclear morphology, chromatin remodeling, and nuclear transport of soluble signaling intermediates using high-resolution fluorescence anisotropy imaging. Using this approach, we show the timescales associated with force-induced polymerization of actin and changes in the F/G actin ratio resulting in nuclear translocation of the G-actin-associated transcriptional cofactor, megakaryoblastic acute leukemia factor-1 (MKL). Further, this method of measuring nuclear organization at high spatiotemporal resolution with simultaneous force application revealed the physical propagation of forces to the nucleus, resulting in changes to chromatin organization, followed by nuclear deformation. We also describe a quantitative model that incorporates active stresses and chemical kinetics to evaluate the observed timescales. Our work suggests that mechanical activation of cells is accompanied by distinct timescales involved in the reorganization of actin and chromatin assembly, followed by translocation of transcription cofactors from the cytoplasm to the nucleus., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

An important goal in studies of protein aggregation is to obtain an understanding of the structural diversity that is characteristic of amyloid fibril and protofibril structures at the molecular level. In this study, what to our knowledge are novel assays based on time-resolved fluorescence anisotropy decay and dynamic quenching measurements of a fluorophore placed at different specific locations in the primary structure of a small protein, barstar, have been used to determine the extent to which the protein sequence participates in the structural core of protofibrils. The fluorescence measurements reveal the structural basis of how modulating solvent polarity results in the tuning of the protofibril conformation from a pair of parallel β-sheets in heat-induced protofibrils to a single parallel β-sheet in trifluorethanol-induced protofibrils. In trifluorethanol-induced protofibrils, the single β-sheet is shown to be built up from in-register β-strands formed by nearly the entire protein sequence, while in heat-induced protofibrils, the pair of β-sheets motif is built up from β-strands formed by only the last two-third of the protein sequence., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (∼1 s) and an unusually stable (∼600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We present a theoretical technique for quantifying the cellular copy-number of fluorophores that relies on the random nature of the photobleaching process. Our approach does not require single-molecule sensitivity, and therefore can be used with commonly used epifluorescence microscopes. Fluctuations arising from photobleaching can be used to estimate the proportionality between fluorescence intensity and copy-number, which can then be used with subsequent intensity measurements to estimate copy-number. We calculate the statistical errors of our approach and verify them with stochastic simulations. By using fluctuations over the entire photobleaching process, we obtain significantly smaller errors than previous approaches that have used fluctuations arising from cytoplasmic proteins partitioning during cellular division. From the time-dependence of the fluctuations as photobleaching proceeds, we can discriminate between desired photobleach fluctuations and background noise or photon shot noise. Our approach does not require cellular division and the photobleaching rate sets a timescale that is adjustable with respect to cellular processes. We hope that our approach will now be applied experimentally., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The synaptic vesicle (SV) is a central organelle in neurotransmission, and previous studies have suggested that SV protein 2 (SV2) may be responsible for forming a gel-like matrix within the vesicle. Here we measured the steady-state rotational anisotropy of the fluorescent dye, Oregon Green, within individual SVs. By also measuring the fluorescence lifetime of Oregon Green in SVs, we determined the mean rotational viscosity to be 16.49 ± 0.12 cP for wild-type (WT) empty mice vesicles (i.e., with no neurotransmitters), 11.21 ± 0.12 cP for empty vesicles from SV2 knock-out mice, and 11.40 ± 0.65 cP for WT mice vesicles loaded with the neurotransmitter glutamate (Glu). This measurement shows that SV2 is an important determinant of viscosity within the vesicle lumen, and that the viscosity decreases when the vesicles are filled with Glu. The viscosities of both empty SV2 knock-out vesicles and Glu-loaded WT vesicles were significantly different from that of empty WT SVs (p < 0.05). This measurement represents the smallest enclosed volume in which rotational viscosity has been measured thus far., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131Δ), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions, Hsp90 partially closes around Δ131Δ, and in the presence of AMPPNP, Δ131Δ binds with increased affinity to Hsp90's fully closed state. FRET measurements show that Δ131Δ accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured region of Δ131Δ. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein, and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The interaction of a melittin mutant with a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-supported lipid bilayer was studied with the use of time-resolved evanescent wave-induced fluorescence spectroscopy (TREWIFS) and evanescent wave-induced time-resolved fluorescence anisotropy measurements (EW-TRAMs). The mutant peptide was labeled at position K14 with AlexaFluor 430 and retained the lytic activity characteristic of native melittin. The fluorescence decay kinetics of the conjugate was found to be biexponential with a short-lived component, τ(1), due to photoinduced electron transfer between AlexaFluor 430 and proximal side chains within or between the peptides. The longer-lived component, τ(2), was sensitive to the polarity of the microenvironment at or near the K14 position of the peptide. Upon interaction with a DPPC-supported bilayer, the proportional contribution of τ(1) increased, indicating a conformational change of the peptide. The values of τ(1) and τ(2) indicate that the AlexaFluor 430 probe experienced an environment with an equivalent polarity no less than that of methanol. EW-TRAMs data from the melittin mutant revealed hindered rotational motions of the AlexaFluor 430 probe both in the plane and perpendicular to the plane of the supported lipid bilayer. The data indicate a highly ordered and polar environment near the center of the melittin helix consistent with the formation of a toroidal pore., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10(-15) L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Netrin receptor DCC plays critical roles in many cellular processes, including axonal outgrowth and migration, angiogenesis, and apoptosis, but the molecular basis of DCC-mediated signaling is largely unclear. ERK2, a member of the MAPK family, is one of the few proteins known to be involved in DCC-mediated signaling. Here, we report that ERK2 directly interacts with DCC, and the ERK2-binding region was mapped to the conserved intracellular P1 domain of the receptor. The structure of ERK2 in complex with the P1 domain of DCC reveals that DCC contains a MAPK docking motif. The docking of the P1 domain onto ERK2 physically positions several phosphorylation sites of DCC in the vicinity of the kinase active site. We further show that the docking interaction between the P1 domain and ERK2 is essential for the ERK2-mediated phosphorylation of DCC. We conclude that DCC signaling is directly coupled with MAPK signaling cascades., (Copyright © 2010 Elsevier Ltd. All rights reserved.)