Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.
AuthorsSlaughter BD, Unruh JR, Price ES, Huynh JL, Bieber Urbauer RJ, Johnson CK
JournalJ Am Chem Soc
PubMed ID16117552
'We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes ... More
An allosteric mechanism controls antigen presentation by the H-2K(b) complex.
AuthorsGakamsky DM, Boyd LF, Margulies DH, Davis DM, Strominger JL, Pecht I
JournalBiochemistry
PubMed ID10508421
'The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were ... More
High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation.
AuthorsWashburn NR, Yamada KM, Simon CG, Kennedy SB, Amis EJ
JournalBiomaterials
PubMed ID14643595
'A high-throughput method for analyzing cellular response to crystallinity in a polymer material is presented. Variations in crystallinity lead to changes in surface roughness on nanometer length scales, and it is shown that cells are exquisitely sensitive to these changes. Gradients of polymer crystallinity were fabricated on films of poly(L-lactic ... More
Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments.
AuthorsAllen MW, Urbauer RJ, Zaidi A, Williams TD, Urbauer JL, Johnson CK
JournalAnal Biochem
PubMed ID14751262
'We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring ... More
Influence of ADP on cross-bridge-dependent activation of myofibrillar thin filaments.
AuthorsZhang D, Yancey KW, Swartz DR
JournalBiophys J
PubMed ID10827987
'Contraction of skeletal muscle is regulated by calcium at the level of the thin filament via troponin and tropomyosin. Studies have indicated that strong cross-bridge binding is also involved in activation of the thin filament. To further test this, myofibrils were incubated with a wide range of fluorescent myosin subfragment ... More
A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA.
AuthorsDodge A, Turcatti G, Lawrence I, de Rooij NF, Verpoorte E
JournalAnal Chem
PubMed ID15018583
'This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the ... More
Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix.
AuthorsReshetnyak YK, Andreev OA, Lehnert U, Engelman DM
JournalProc Natl Acad Sci U S A
PubMed ID16608910
'We have previously observed the spontaneous, pH-dependent insertion of a water-soluble peptide to form a helix across lipid bilayers [Hunt, J. F., Rath, P., Rothschild, K. J. & Engelman, D. M. (1997) Biochemistry 36, 15177-15192]. We now use a related peptide, pH (low) insertion peptide, to translocate cargo molecules attached ... More
Site-specific fluorescence labelling of recombinant polyomavirus-like particles.
AuthorsSchmidt U, Kenklies J, Rudolph R, Böhm G
JournalBiol Chem
PubMed ID10223344
For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas ... More
Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts.
AuthorsHalter M, Tona A, Bhadriraju K, Plant AL, Elliott JT,
JournalCytometry A
PubMed ID17828790
To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 ... More
Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy.
AuthorsBhadriraju K, Elliott JT, Nguyen M, Plant AL,
JournalBMC Cell Biol
PubMed ID17941977
BACKGROUND: In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation ... More
Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles.
AuthorsDe Keyzer J, Van Der Does C, Driessen AJ
JournalJ Biol Chem
PubMed ID12226104
Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, ... More
Potentiation of chlorin e6 photodynamic activity in vitro with peptide-based intracellular vehicles.
AuthorsBisland SK, Singh D, Gariépy J
JournalBioconjug Chem
PubMed ID10563767
Photodynamic therapy (PDT) is a targeted treatment modality where photosensitizers accumulate into cells and are selectively activated by light leading to the production of toxic species and cell death. Focusing the action of photosensitizers to a unique intracellular target may enhance their cytotoxicity. In this study, we demonstrate that the ... More
Binding of the b-subunit in the ATP synthase from Escherichia coli.
AuthorsDiez M, Börsch M, Zimmermann B, Turina P, Dunn SD, Gräber P
JournalBiochemistry
PubMed ID14744151
The rotary mechanism of ATP synthase requires a strong binding within stator subunits. In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant ... More
Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins.
AuthorsGambhir A, Hangyás-Mihályné G, Zaitseva I, Cafiso DS, Wang J, Murray D, Pentyala SN, Smith SO, McLaughlin S
JournalBiophys J
PubMed ID15041659
The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of ... More
Trafficking of spontaneously endocytosed MHC proteins.
AuthorsChiu I, Davis DM, Strominger JL
JournalProc Natl Acad Sci U S A
PubMed ID10570178
Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. beta(2)-microglobulin (beta(2)m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell ... More
Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification.
AuthorsSlaughter BD, Unruh JR, Allen MW, Bieber Urbauer RJ, Johnson CK
JournalBiochemistry
PubMed ID15751946
A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains ... More