RESUMO
Magnetic tweezers permit application of precisely calibrated stretching forces to nucleic acid molecules tethered between a surface and superparamagnetic beads. In addition, magnetic tweezers can control the tethers' twist. Here, we focus on recent extensions of the technique that expand the capabilities of conventional magnetic tweezers by enabling direct measurements of single-molecule torque and twist. Magnetic torque tweezers (MTT) still control the DNA or RNA tether's twist, but directly measure molecular torque by monitoring changes in the equilibrium rotation angle upon overwinding and underwinding of the tether. In freely orbiting magnetic tweezers (FOMT), one end of the tether is allowed to rotate freely, while still applying stretching forces and monitoring rotation angle. Both MTT and FOMT have provided unique insights into the mechanical properties, structural transitions, and interactions of DNA and RNA. Here, we provide step-by-step protocols to carry out FOMT and MTT measurements. In particular, we focus on multiplexed measurements, i.e., measurements that record data for multiple nucleic acid tethers at the same time, to improve statistics and to facilitate the observation of rare events.
Assuntos
Magnetismo/métodos , Pinças Ópticas , Imagem Individual de Molécula , Calibragem , DNA/análise , Campos Magnéticos , Microesferas , Soluções , TorqueRESUMO
We have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 using the acute promyelocytic leukemia cell line NB4 together with granulocyte colony-stimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27Kip1 accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27Kip1 dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27Kip1 confirmed that tyrosine-phosphorylated p27Kip1 preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27Kip1 point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27Kip1. Taken together, this report provides the first evidence that tyrosine phosphorylation of p27Kip1 plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF.
Assuntos
Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Fosforilação , Ligação Proteica , Tirosina/metabolismoRESUMO
A genetically modified form of the human DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.
Assuntos
Microscopia de Força Atômica/métodos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sítios de Ligação , Conectina , Reparo do DNA , Vidro/química , Glutationa Transferase/metabolismo , Ouro/química , Humanos , Imunoglobulinas/química , Proteínas Musculares/química , O(6)-Metilguanina-DNA Metiltransferase/genética , Proteínas Quinases/química , Proteínas Recombinantes de Fusão/genética , Espectrometria de Fluorescência/métodos , Ressonância de Plasmônio de Superfície/métodos , Sequências de Repetição em Tandem , Fatores de TempoRESUMO
Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.
