ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.

The tetracysteine (tc) tag/biarsenical dye system (FlAsH or ReAsH) promises to combine the flexibility of fluorescent protein tags with the small size of dye labels, allowing in-cell study of target proteins that are perturbed by large protein tags. Quantitative thermodynamic and kinetic studies in-cell using FlAsH and ReAsH have been hampered by methodological complexities presented by the fluorescence properties of the tag-dye complex probed by either Förster resonance energy transfer (FRET) or direct excitation. We label the model protein phosphoglycerate kinase (PGK) with AcGFP1 and ReAsH for direct comparison with AcGFP1/mCherry-labeled PGK. We find that fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells. We also demonstrate the to us surprising result that fluorescence from directly excited, unburied ReAsH at the C-terminus of the model protein also reports on folding in vitro and in cells. Comparing the ReAsH-labeled protein to a construct labeled with two fluorescent protein tags allows us to evaluate how a bulkier protein tag affects protein dynamics in cells and in vitro. We find that the average folding rate in the cell is closer to the in vitro rate with the smaller tag, highlighting the effect of tags on quantitative in-cell measurements.

Misplaced helix slows down ultrafast pressure-jump protein folding.

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 µs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 µs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little ß-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.

Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations.

The unimolecular folding reaction of small proteins is now amenable to a very direct mechanistic comparison between experiment and simulation. We present such a comparison of microsecond pressure and temperature jump refolding kinetics of the engineered WW domain FiP35, a model system for ß-sheet folding. Both perturbations produce experimentally a faster and a slower kinetic phase, and the "slow" microsecond phase is activated. The fast phase shows differences between perturbation methods and is closer to the downhill limit by temperature jump, but closer to the transiently populated intermediate limit by pressure jump. These observations make more demands on simulations of the folding process than just a rough comparison of time scales. To complement experiments, we carried out several pressure jump and temperature jump all-atom molecular dynamics trajectories in explicit solvent, where FiP35 folded in five of the six simulations. We analyzed our pressure jump simulations by kinetic modeling and found that the pressure jump experiments and MD simulations are most consistent with a 4-state kinetic mechanism. Together, our experimental and computational data highlight FiP35's position at the boundary where activated intermediates and downhill folding meet, and we show that this model protein is an excellent candidate for further pressure jump molecular dynamics studies to compare experiment and modeling at the folding mechanism level.

Quinary protein structure and the consequences of crowding in living cells: leaving the test-tube behind.

Although the importance of weak protein-protein interactions has been understood since the 1980s, scant attention has been paid to this "quinary structure". The transient nature of quinary structure facilitates dynamic sub-cellular organization through loose grouping of proteins with multiple binding partners. Despite our growing appreciation of the quinary structure paradigm in cell biology, we do not yet understand how the many forces inside the cell--the excluded volume effect, the "stickiness" of the cytoplasm, and hydrodynamic interactions--perturb the weakest functional protein interactions. We discuss the unresolved problem of how the forces in the cell modulate quinary structure, and to what extent the cell has evolved to exert control over the weakest biomolecular interactions. We conclude by highlighting the new experimental and computational tools coming on-line for in vivo studies, which are a critical next step if we are to understand quinary structure in its native environment.

Temporal variation of a protein folding energy landscape in the cell.

Chemical reaction rate coefficients and free energies are usually time-independent quantities. Protein folding in vitro is one such reaction with a fixed energy landscape. However, in the milieu of the cell, the energy landscape can be modulated in space and time by fluctuations in the intracellular environment such as cytoskeletal rearrangements, changes in biomolecule concentrations, and large scale cellular reorganization. We studied the time dependence of the folding landscape of a FRET-labeled enzyme, yeast phosphoglycerate kinase (PGK-FRET). Living U2OS cells served as our test tube, and the mammalian cell cycle, a process strictly regulated in time, served as our clock. We found that both the rate of folding and the thermodynamic stability of PGK-FRET are cell cycle-dependent. We also assayed folding rates of PGK-FRET in spatial proximity to and far away from mitotic chromosomes. Our results show that expedited folding in DNA-rich regions cannot account for the faster rate of PGK-FRET folding in mitotic cells.

Fluorescein-labeled glutathione to study protein S-glutathionylation.

Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins.

Critical phenomena in the temperature-pressure-crowding phase diagram of a protein.

In the cell, proteins fold and perform complex functions through global structural rearrangements. Function requires a protein to be at the brink of stability to be susceptible to small environmental fluctuations, yet stable enough to maintain structural integrity. These apparently conflicting behaviors are exhibited by systems near a critical point, where distinct phases merge-a concept beyond previous studies indicating proteins have a well-defined folded/unfolded phase boundary in the pressure-temperature plane. Here, by modeling the protein phosphoglycerate kinase (PGK) on the temperature (T), pressure (P), and crowding volume-fraction (ϕ) phase diagram, we demonstrate a critical transition where phases merge, and PGK exhibits large structural fluctuations. Above the critical point, the difference between the intermediate and unfolded phases disappears. When ϕ increases, the critical point moves to lower T c. We verify the calculations with experiments mapping the T-P-ϕ space, which likewise reveal a critical point at 305 K and 170 MPa that moves to lower T c as ϕ increases. Crowding places PGK near a critical line in its natural parameter space, where large conformational changes can occur without costly free energy barriers. Specific structures are proposed for each phase based on simulation.

The Gold Nanorod-Biology Interface: From Proteins to Cells to Tissue.

Gold nanorods absorb and scatter light strongly in the near-infrared portion of the electromagnetic spectrum, making them ideal tissue contrast agents for imaging techniques such as optical coherence tomography (OCT). Strong interactions occur at the nano-bio interface, such as proteins binding to gold nanorods forming a 'corona.' To fulfill the promise of nanorods for applications such as contrast agents, we must better understand the intrinsic interactions of these nanomaterials with biological systems at the molecular, cellular and tissue level. In this paper, we briefly review the nanorod-protein interface. We then present some new fast relaxation imaging (FReI) measurements of how the presence of strongly-absorbing gold nanorods affects protein binding and folding, taking into account inner filter effects and the strong quenching effect of nanorods on fluorescent-labeled proteins. Next we show that two-photon photoluminescence of the gold nanorods can be used to image the nanorods in tissue constructs, allowing us to independently study their tissue distribution so they can be used successfully as contrast agents in optical coherence microscopy.