RESUMO
Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of gamma-COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single-pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in gamma-COP is transmitted within the complex to its alpha-subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.
Assuntos
Proteína Coatomer/química , Proteína Coatomer/metabolismo , Conformação Proteica , Animais , Proteína Coatomer/genética , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Complexo de Golgi/metabolismo , Ligantes , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , CoelhosRESUMO
We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.
Assuntos
DNA/química , Lasers , Microscopia Confocal/métodos , Termodinâmica , Cor , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Dispositivos Ópticos , Sensibilidade e EspecificidadeRESUMO
Current efforts to monitor the diffusion of proteins in living cells are based on either fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching, or image correlation spectroscopy. However, these methods cannot generate a map of diffusion times. Here, we introduce a new method termed diffusion imaging microscopy that combines scanning confocal microscopy, time-correlated single-photon counting, and FCS and thus allows us to measure spatially resolved diffusion times. In our approach, we record scan images with time-resolved photon streams within each individual pixel. By extending the pixel dwell time to 25-100 ms, a software correlation of individual photons within each pixel yields the average diffusion time. Additionally, information on fluorescence intensity (number of photons) and fluorescence lifetime is available and can be used to sort fluorescence photons and to discriminate from autofluorescence. We evaluated our method by measuring diffusion times of dT20-TMR in solutions of different viscosity. We further demonstrate the applicability of the method to living cells and recorded a diffusion map of a living 3T3 mouse fibroblast incubated with dT20-ATTO488.