Chaperones Hsc70 and Hsp70 Bind to the Protein PGK Differently inside Living Cells.

Differences in the physical interactions between proteins, such as binding equilibria, can provide clues about the differences in their function. The binding of heat shock proteins to substrate proteins in living cells is one such example. Eukaryotic cells have evolved many homologues in the Hsp70 family of heat shock proteins, each of which is specialized for a specific function. We previously showed that Hsp70, which is upregulated during heat shock, binds to the model substrate phosphoglycerate kinase (PGK) in human cells before PGK completely unfolds. We dubbed this the "preemptive holding" mechanism. Here, we studied the homologue Hsc70 (heat shock cognate protein), which is constitutively expressed in human cells even in the absence of heat shock. Recent literature has demonstrated the multiple functions performed by Hsc70 in cells under normal conditions. Despite the name "heat shock cognate", very few studies have shown whether Hsc70 is actually involved in the heat shock response. Here we corroborate the existence of the in-cell heat shock response of Hsc70. We show that Hsc70 binds to PGK in human cells in a cooperative manner that directly correlates with protein thermal unfolding. This "unfolded state holding" mechanism differs from the Hsp70 "preemptive holding" mechanism. We rationalize this difference by protein evolution; unlike Hsp70, which is upregulated in order to bind proteins specifically during heat shock, the finite amount of Hsc70 in cells cannot afford to bind to still-folded proteins without compromising its multiple other functions.

Heat shock-induced chaperoning by Hsp70 is enabled in-cell.

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard ß-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a "preemptive holdase" mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

Weak Chemical Interactions That Drive Protein Evolution: Crowding, Sticking, and Quinary Structure in Folding and Function.

In recent years, better instrumentation and greater computing power have enabled the imaging of elusive biomolecule dynamics in cells, driving many advances in understanding the chemical organization of biological systems. The focus of this Review is on interactions in the cell that affect both biomolecular stability and function and modulate them. The same protein or nucleic acid can behave differently depending on the time in the cell cycle, the location in a specific compartment, or the stresses acting on the cell. We describe in detail the crowding, sticking, and quinary structure in the cell and the current methods to quantify them both in vitro and in vivo. Finally, we discuss protein evolution in the cell in light of current biophysical evidence. We describe the factors that drive protein evolution and shape protein interaction networks. These interactions can significantly affect the free energy, ΔG, of marginally stable and low-population proteins and, due to epistasis, direct the evolutionary pathways in an organism. We finally conclude by providing an outlook on experiments to come and the possibility of collaborative evolutionary biology and biophysical efforts.

Dodine as a Kosmo-Chaotropic Agent.

Denaturants such as the guanidinium cation unfold proteins at molar concentrations, which interferes with ultraviolet- and infrared-based spectroscopy measurements. Dodine denatures some proteins cooperatively at a thousand-fold lower concentration, allowing for spectroscopy measurements. Nonetheless, dodine's microscopic mechanism of interaction with proteins is not understood. We probe the effect of dodine on α-helices and tertiary structure by investigating the stability of the small helical protein B. Experiments show that dodine promotes formation of helical structure (a kosmotropic effect), while inducing the loss of tertiary structure (a chaotropic effect). Although dodine destabilizes native protein structure, it does not lower the thermal denaturation midpoint temperature of protein B. All-atom simulations reveal the cause for both observations: The denaturant action of dodine's guanidyl headgroup is counteracted by its aliphatic tail, which stabilizes amphipathic helices and associates with an expanded protein core. The Janus-like behavior of headgroup and tail make dodine a simultaneous stabilizer-destabilizer or "kosmo-chaotrope".

Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins.

The widespread interest in neutral, water-soluble polymers such as poly(ethylene glycol) (PEG) and poly(zwitterions) such as poly(sulfobetaine) (pSB) for biomedical applications is due to their widely assumed low protein binding. Here we demonstrate that pSB chains in solution can interact with proteins directly. Moreover, pSB can reduce the thermal stability and increase the protein folding cooperativity relative to proteins in buffer or in PEG solutions. Polymer-dependent changes in the tryptophan fluorescence spectra of three structurally-distinct proteins reveal that soluble, 100 kDa pSB interacts directly with all three proteins and changes both the local polarity near tryptophan residues and the protein conformation. Thermal denaturation studies show that the protein melting temperatures decrease by as much as ∼1.9 °C per weight percent of polymer and that protein folding cooperativity increases by as much as ∼130 J mol-1 K-1 per weight percent of polymer. The exact extent of the changes is protein-dependent, as some proteins exhibit increased stability, whereas others experience decreased stability at high soluble pSB concentrations. These results suggest that pSB is not universally protein-repellent and that its efficacy in biotechnological applications will depend on the specific proteins used.

Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels.

We apply fast relaxation imaging (FReI) as a novel technique for investigating the folding stability and dynamics of proteins within polyacrylamide hydrogels, which have diverse and widespread uses in biotechnology. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after temperature jump perturbations. Unlike bulk measurements, diffraction-limited epifluorescence imaging combined with fast temperature perturbations reveals the impact of local environment effects on protein-biomaterial compatibility. Our experiments investigated a crowding sensor protein (CrH2) and phosphoglycerate kinase (PGK), which undergoes cooperative unfolding. The crowding sensor quantifies the confinement effect of the cross-linked hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution (no confinement), while the 10% hydrogel is strongly confining. FRAP measurements and protein concentration gradients in the 4% and 10% hydrogels further support this observation. PGK reveals that noncovalent interactions of the protein with the polymer surface are more important than confinement for determining protein properties in the gel: the mere presence of hydrogel increases protein stability, speeds up folding relaxation, and promotes irreversible binding to the polymer even at the solution-gel interface, whereas the difference between the 4% and the 10% hydrogels is negligible despite their large difference in confinement. The imaging capabilities of FReI, demonstrated to be diffraction limited, further revealed spatially homogeneous protein unfolding across the hydrogels at 500 nm length scales and revealed differences in protein properties at the gel-solution boundary.

The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive.

It is frequently assumed that fluorescent protein tags used in biological imaging experiments are minimally perturbing to their host protein. As in-cell experiments become more quantitative and measure rates and equilibrium constants, rather than just "on-off" activity or the presence of a protein, it becomes more important to understand such perturbations. One criterion for a protein modification to be a perturbation is additivity of two perturbations (a linear effect on the protein free energy). Here we show that adding fluorescent protein tags to a host protein in vitro has a large nonadditive effect on its folding free energy. We compare an unlabeled, three singly labeled, and a doubly labeled enzyme (phosphoglycerate kinase). We propose two mechanisms for nonadditivity. In the "quinary interaction" mechanism, two tags interact transiently with one another, relieving the host protein from unfavorable tag-protein interactions. In the "crowding" mechanism, adding two tags provides the minimal crowding necessary to overcome destabilizing interactions of individual tags with the host protein. Both of these mechanisms affect protein stability in cells; we show here that they must also be considered for tagged proteins used for reference in vitro.

Dodine as a transparent protein denaturant for circular dichroism and infrared studies.

The fungicide dodine combines the cooperative denaturation properties of guanidine with the mM denaturation activity of SDS. It was previously tested only on two small model proteins. Here we show that it can be used as a chemical denaturant for phosphoglycerate kinase (PGK), a much larger two-domain enzyme. In addition to its properties as a chemical denaturant, dodine facilitates thermal denaturation of PGK, and we show for the first time that it also facilitates pressure denaturation of a protein. Much higher quality circular dichroism and amide I' infrared spectra of PGK can be obtained in dodine than in guanidine, opening the possibility for use of dodine as a denaturant when UV or IR detection is desirable. One caution is that dodine denaturation, like other detergent-based denaturants, is less reversible than guanidine denaturation.