Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

We characterized the effect of the first basepair on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of double-stranded DNA using gaussian-mixture adaptive umbrella sampling simulations. In the simulations, the sampling of all five dihedral angles along the linker was enhanced, so that both stacked and unstacked states were sampled. The affinity of Cy3 for a T·A basepair (with the dye attached to T) was found to be significantly less than for the other basepairs. This was verified experimentally by measuring the activation energies for cis-trans isomerization of the dye. The simulation and experimental results indicate the existence of partially unstacked conformations amenable to photoisomerization. The simulations also showed that stacking of Cy3 straightens the DNA while stabilizing the first basepair. Our findings indicate that fluorescence is modulated by Cy3-DNA interactions in a sequence-dependent manner.

Sliding clamps are ring-shaped oligomeric proteins that are essential for processive deoxyribonucleic acid replication. Although crystallographic structures of several clamps have been determined, much less is known about clamp structure and dynamics in solution. Here, we characterized the intrinsic solution stability and oligomerization dynamics of the homodimeric Escherichia coli β and the homotrimeric Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) clamps using single-molecule approaches. We show that E. coli β is stable in solution as a closed ring at concentrations three orders of magnitude lower than PCNA. The trimeric structure of PCNA results in slow subunit association rates and is largely responsible for the lower solution stability. Despite this large difference, the intrinsic lifetimes of the rings differ by only one order of magnitude. Our results show that the longer lifetime of the E. coli β dimer is due to more prominent electrostatic interactions that stabilize the subunit interfaces.

The long-term goal of this project is to design complex membrane proteins that have been functionalized with appropriate cofactors and can serve as components of artificial bioenergetic systems. We seek to achieve this by engineering specific interactions between the helical interfaces of transmembrane proteins. This will allow us to create functional nanostructures that contain the appropriate components in particular orientations. The association of transmembrane helical proteins to homodimers is driven by a handful of sequence motifs, the most common of which is GXXXG. We used this sequence to introduce additional interactions so that the formation of heterodimers occurred.We designed a buried salt bridge in the transmembrane domain of a well known dimeric membrane protein, GpA. We mutated Thr 87, which is not part of the dimerization interface, to diaminopropionic acid (Dap) on one of the helices and to aspartic acid on the other. Dap and aspartic acid interact electrostatically only in a narrow pH window. We characterized the pHdependent association of the peptides when incorporated into micelles using fluorescence resonance energy transfer (FRET). This allowed us to establish the pH profile for heterodimer formation and to measure the strength of the interaction.

2016-12-23T18:47:23