Unique Features of the Folding Landscape of a Repeat Protein Revealed by Pressure Perturbation

Proteins denature not only at high, but also at low temperature as well as high pressure. These denatured states are not easily accessible for experiment, because usually heat denaturation causes aggregation, whereas cold or pressure denaturation occurs at temperatures well below the freezing point of water or pressures above 5 kbar, respectively. Here we have obtained atomic details of the pressure-assisted, cold-denatured state of ubiquitin at 2,500 bar and 258 K by high-resolution NMR techniques. Under these conditions, a folded, native-like and a disordered state exist in slow exchange. Secondary chemical shifts show that the disordered state has structural propensities for a native-like N-terminal β-hairpin and α-helix and a nonnative C-terminal α-helix. These propensities are very similar to the previously described alcohol-denatured (A-)state. Similar to the A-state, 15N relaxation data indicate that the secondary structure elements move as independent segments. The close similarity of pressure-assisted, cold-denatured, and alcohol-denatured states with native and nonnative secondary elements supports a hierarchical mechanism of folding and supports the notion that similar to alcohol, pressure and cold reduce the hydrophobic effect. Indeed, at nondenaturing concentrations of methanol, a complete transition from the native to the A-state can be achieved at ambient temperature by varying the pressure from 1 to 2,500 bar. The methanol-assisted pressure transition is completely reversible and can also be induced in protein G. This method should allow highly detailed studies of protein-folding transitions in a continuous and reversible manner.protein unfolding | thermodynamics | protein dynamics | heteronuclear NMR I t has long been known that proteins unfold not only at high temperatures, but also at high pressures (1) as well as low temperatures (2). The so-called heat, pressure, and cold denaturations can be described in a unified way by Hawley's theory (1,3). This theory assumes a simplified two-state model of protein unfolding, where the free energy difference between folded and unfolded states is a general parabolic function of temperature and pressure:In Eq. 1 Δ indicates the difference of the respective value between the denatured and the native state; β is the compressibility factor ð∂V =∂ pÞ T ; α is the thermal expansivity factor ð∂V =∂TÞ p ; C p is the heat capacity Tð∂S=∂TÞ p ; and p 0 , T 0 is an arbitrarily chosen reference point. The phase boundary between denatured and native states is then given by the condition ΔG = 0, which corresponds to a tilted ellipse within the pT plane for commonly observed values of Δβ < 0, ΔC p > 0, and Δα > 0. This two-state model is clearly an oversimplification, because both folded and unfolded states can be heterogeneous, and due to the paucity of data it is also unclear whether the heat-, cold-, and pressuredenatured states are identical. Nevertheless the model presents a valuable general framework to pinpoint the main contributing thermodynamic entities, which...

Section: Discussionmentioning

confidence: 90%

High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin

Vajpai

Nisius

Wiktor

et al. 2013

“…Rather, increasing pressure destabilizes the tertiary structure of proteins, which, in turn, destabilizes helical structures due to a cooperative destabilization of the folded state. Equilibrium intermediates with residual helical structure have frequently been observed in pressure-induced unfolding experiments monitored by NMR (19,(46)(47)(48). The population of these persisting helical structure can be explained by the opposing effects of pressure on the stability of tertiary and helical structure.…”

Section: Discussionmentioning

confidence: 99%

Transition state and ground state properties of the helix–coil transition in peptides deduced from high-pressure studies

Neumaier

Büttner

Bachmann

et al. 2013

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet-triplet energy transfer at high pressure to determine volume changes associated with the helix-coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressureunfolded proteins. Both helix folding and unfolding become slower with increasing pressure. per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.protein stability | helix dynamics | protein dynamics U nderstanding the effect of pressure on protein stability and dynamics provides insight into fundamental principles and mechanisms of protein folding (1). For most proteins, increasing pressure shifts the folding equilibrium toward the unfolded state, which, according to Le Chatelier's principle, shows that the native state has a larger volume than the unfolded state (2-4). The origin of the volume increase upon folding has been discussed controversially for a long time. The major obstacle in the interpretation of volume changes is opposing contributions from different effects. Analysis of high-resolution X-ray structures suggested that formation of intramolecular hydrogen bonds and van der Waals interactions in the native state lead to a decrease in atomic volumes and thus to a decrease in protein volume upon folding (5). Further, water around hydrophobic groups has a larger volume than bulk water, which also leads to a decrease in volume upon burial of hydrophobic groups in the native protein (6). On the other hand, formation of ordered water structures around charged groups (7) and solvation of the peptide backbone decrease the water volume (6), which leads to a volume increase upon folding. Recent experimental results suggested that volume changes associated with transfer of groups from solvent to the protein interior upon folding are small and that the formation of void volumes in native proteins is the major origin of the volume increase upon folding (8).Volume changes associated with the formation of protein folding transition states are only poorly characterized. Highpressure stopped-flow experiments on tendamistat (9) and cold shock protein (10), as well as pressure-jump experiments on coldshock protein (10) and an ankyrin repeat domain (11), revealed that volumes of protein folding transition states are close to the volume of the native...

“…Folded proteins have a larger partial molar volume than pressure-denatured proteins (by about 10 1 -10 2 mL/mol) (6). The fractal dimension of their folded state is less than 3 because voids occur whenever a connected chain made from a finite amino acid alphabet is packed into a compact structure (7,8).…”

mentioning

confidence: 99%

Misplaced helix slows down ultrafast pressure-jump protein folding

Prigozhin¹,

Liu²,

Wirth³

et al. 2013

Self Cite

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-coilrich 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-jumpinduced 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 β-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.downhill folding | fluorescence lifetime | molecular dynamics simulation | thermal denaturation | lambda repressor T emperature and pressure are excellent perturbations when comparing experimental folding kinetics with molecular dynamics (MD) simulations (1). Fast temperature-jumps (T-jumps) and pressure-jumps (P-jumps) are relatively easy to simulate by MD. Typical temperature changes required to cross the protein folding transition are 5-20 K, easily implemented with laser T-jumps (2). Typical pressure changes required to cross the folding transition are 1-5 kbar, previously achieved only with millisecond time resolution (piezo methods are limited to ΔP < 100 bar) (3, 4). We recently reported a P-jump instrument capable of >1-kbar P-drops with ∼1-μs dead time (5).Folded proteins have a larger partial molar volume than pressure-denatured proteins (by about 10 1 -10 2 mL/mol) (6). The fractal dimension of their folded state is less than 3 because voids occur whenever a connected chain made from a finite amino acid alphabet is packed into a compact structure (7,8). Such imperfections in packing, which disappear when small water molecules solvate the polypeptide chain, lead to protein unfolding under pressure (9). Pressure unfolding is a slow process because the positive activation volume is unfavorable at high pressure (10).Here, we study the much faster process of protein refolding at 1 bar and room temperature, starting from the pressure-d...