Biomedical subjects
Publications and source records attributed to Martin Gruebele.
Proteins can be redesigned to fold downhill on a free energy surface characterized by only a few coordinates, confirming a principal prediction of the 'energy-landscape' model. Nonetheless, natural proteins have small but significant barriers. Spectroscopy and kinetics reveal potential biological causes for activation barriers during protein folding: evolution against protein aggregation and for protein function.
Heat- and cold-denatured proteins are considered separate thermodynamic states because temperature tuning requires the protein to pass through two 'soft' first-order phase transitions. When both pressure and temperature changes are allowed, the heat- and cold-denatured states of proteins can be interconverted without going through the native state. This raises the question of whether these states are distinguished from one another by their folding kinetics. For the Tyr22Trp/Ala37Gly/Ala49Gly mutant of the 80 residue five-helix bundle protein lambda(6-85), we show that viscosity-corrected folding rates do not distinguish the cold- and heat-denatured states. We attribute this to a folding mechanism dominated by hydrophobic collapse. Our 'temperature-symmetric' approach offers an alternative to viscosity tuning with solvent additives in such cases.
The Y22W/Q33Y/G46,48A mutant of the protein lambda6-85 folds in a few microseconds at room temperature. We find that its folding kinetics are probe-dependent under a strong bias toward the native state, a new signature for downhill folding. The IR- and fluorescence-detected relaxation time scales converge when the native bias is removed by raising the temperature, recovering activated two-state folding. Langevin dynamics simulations on one- and 2D free energy surfaces tunable from two-state to downhill folding reproduce the difference between the IR and fluorescence experiments, as well as the temperature and viscosity trends. In addition, the 2D surface reproduces the stretched exponential dynamics that we fit to the glucose solution experimental data at short times. Nonexponential dynamics at <10 micros is a signature either for local free energy minima along the reaction coordinate ("longitudinal roughness"), or for folding on a higher-dimensional free energy surface ("transverse roughness").
Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Here, we demonstrate that an RNA hairpin containing the nucleotide analogue 8-bromoguanosine (8BrG or G), gcUUCGgc, has enhanced stability relative to the unmodified hairpin, with DeltaDeltaG(37)(degrees)= -0.69 +/- 0.15 kcal mol(-1) and DeltaT(M) = +6.8 +/- 1.4 degrees C. NMR spectroscopic data suggest that the enhanced stability of gcUUCGgc does not arise from the native state; laser temperature-jump experiments support this notion, as gcUUCGgc and gcUUCGgc have similar unfolding rate constants, but the folding rate constant of gcUUCGgc is 4.1-fold faster at 37.5 degrees C and 2.8-fold faster under isoenergetic conditions. On the basis of these findings, we propose that 8BrG reduces the conformational entropy of the denatured state, resulting in an accelerated conformational search for the native state and enhanced stability.
Two classes of lambda(6-85) mutants (those richer in alanine, and those richer in glycine) have very similar slopes in an Arrhenius plot of the unfolding rates but very different temperature dependencies of the folding rates. Temperature-dependent interactions (e.g., hydrophobicity) play a large role in the initial stages of folding but not in the initial stages of unfolding of lambda(6-85). Placement of the transition state in terms of its surface exposure and entropy shows that at least two reaction coordinates are required to describe folding of all mutants over the full temperature range. The unusual Arrhenius plots of the very fastest mutant provide an additional kinetic signature for downhill folding.
Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small beta-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a beta-sheet protein.
The folding landscapes of polypeptides and proteins exhibit a hierarchy of local minima. The causes range from proline isomerization all the way down to microstructure in the free energy caused by residual frustration inherent in even the best 20 amino acid design. The corresponding time scales range from hours to submicroseconds. The smallest microstructures are difficult to detect. We have measured the folding/unfolding kinetics of the engineered trpzip2 peptide at different tryptophan fluorescence wavelengths, each yielding a different rate. Wavelength-dependent folding kinetics on 0.1-2 mus time scales show that different microstructures with a range of solvent exposure and local dynamics are populated. We estimate a lower limit for the roughness of the free energy surface based on the range of rates observed.
The beta-hairpin trpzip2 can be tuned continuously from a two-state folder to folding on a rough energy landscape without a dominant refolding barrier. At high denaturant concentration, this extremely stable peptide exhibits a single apparent "two-state" transition temperature when monitored by different spectroscopic techniques. However, under optimal folding conditions the hairpin undergoes an unusual folding process with three clusters of melting transitions ranging from 15 degrees C to 160 degrees C, as monitored by 12 different experimental and computational observables. We explain this behavior in terms of a rough free energy landscape of the unfolded peptide caused by multiple tryptophan interactions and alternative backbone conformations. The landscape is mapped out by potentials of mean force derived from replica-exchange molecular dynamics simulations. Implications for deducing cooperativity from denaturant titrations, for the origin of folding cooperativity, and for the folding of thermophilic proteins are pointed out. trpzip is an excellent small tunable model system for the glass-like folding transitions predicted by landscape theory.
We show that the five-helix bundle lambda(6-85) can be engineered and solvent-tuned to make the transition from activated two-state folding to downhill folding. The transition manifests itself as the appearance of additional dynamics faster than the activated kinetics, followed by the disappearance of the activated kinetics when the bias toward the native state is increased. Our fastest value of 1 micros for the "speed" limit of lambda(6-85) is measured at low concentrations of a denaturant that smooths the free-energy surface. Complete disappearance of the activated phase is obtained in stabilizing glucose buffer. Langevin dynamics on a rough free-energy surface with variable bias toward the native state provides a robust and quantitative description of the transition from activated to downhill folding. Based on our simulation, we estimate the residual energetic frustration of lambda(6-85) to be delta(2) G approximately 0.64 k(2)T(2). We show that lambda(6-86), as well as very fast folding proteins or folding intermediates estimated to lie near the speed limit, provide a better rate-topology correlation than proteins with larger energetic frustration. A limit of beta > or = 0.7 on any stretching of lambda(6-85) barrier-free dynamics suggests that a low-dimensional free-energy surface is sufficient to describe folding.
At room temperature, natural polypeptides exposed to high concentrations of a strong denaturant nearly attain the circular dichroism spectra characteristic of random coils. As temperature is increased, the spectra begin to show the signature of a substantial fraction of extended chain, the structure common in beta-sheets. This structural propensity at high temperature is not altered by concentration changes over a greater than 1000-fold range, so it is not caused by aggregation. Four proteins with different folds and varying amounts of alpha-helical and beta-sheet secondary structure, in the presence or absence of denaturant, all were subject to extended chain formation upon heating. This effect arises naturally from the steric constraints associated with polypeptides and is probably counteracted, not enhanced, by hydrophobic interactions. Molecular dynamics simulations in the 298-1000 K range reveal an attractive potential of mean force in the extended chain region of the Ramachandran diagram, which broadens as the temperature is raised. We also demonstrate a direct correlation between extended structure content and the rate of aggregation kinetics. Thus pre-existing extended structure could funnel proteins into aggregates.
We recently reported stretched kinetics during the formation of a collapsed, long-lived intermediate state of the large two-domain enzyme phosphoglycerate kinase (PGK). It was postulated that intrinsic roughness of the energy landscape on the way downhill to the intermediate causes the lack of a single time-scale. Here, we investigate several alternative explanations for stretched refolding dynamics in more detail: tyrosine fluorescence, multiple tryptophan probes, and rate differences between independently folding domains. To this end, we systematically simplify PGK in several steps from the full protein with two tryptophan residues and all tyrosine residues probed, to a single domain with only one tryptophan residue and no tyrosine residue probed. The kinetics in the 10 micros to 10 ms range are revealed by laser-induced temperature-jump relaxation experiments. The isolated N-terminal domain forms an intermediate by nearly single-exponential kinetics, but the isolated C-terminal domain shows strongly non-exponential kinetics. Thus, domain interaction and a cis-proline residue between the two domains are ruled out as the sole contributors to heterogeneity during the earliest folding dynamics of the C-terminal domain. We apply two limiting models for the roughness of the energy landscape. A sequential three-state model lumps all the roughness into a single trap. The "strange kinetics" model with logarithmic oscillations developed by Klafter and co-workers distributes the roughness over a larger number of states. Both models explain our data about equally well, but the coincidental values of rate constants in all of our double-exponential fits, and the absence of a spectroscopic signature distinct from the endpoints of the folding process favors more roughness than can be explained by just a single trap.
Many small proteins seem to fold by a simple process explicable by conventional chemical kinetics and transition-state theory. This assumes an instant equilibrium between reactants and a high-energy activated state. In reality, equilibration occurs on timescales dependent on the molecules involved, below which such analyses break down. The molecular timescale, normally too short to be seen in experiments, can be of a significant length for proteins. To probe it directly, we studied very rapidly folding mutants of the five-helix bundle protein lambda(6-85), whose activated state is significantly populated during folding. A time-dependent rate coefficient below 2 micro s signals the onset of the molecular timescale, and hence the ultimate speed limit for folding. A simple model shows that the molecular timescale represents the natural pre-factor for transition state models of folding.
The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded beta-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28-37 residues), the availability of a quantitative reaction coordinate (phi(T)), the fast folding time scale (10s of micros), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory.
Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10-100 min) and very fast (4 micro s-2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales.
For the first time, data are presented on the time-resolved disassembly reaction of a highly organized membrane protein complex in vitro. The photosynthetic core light-harvesting complex of the bacterial strain Rhodospirillum rubrum G9 consists of 12-16 dimeric subunits that in vivo are associated with the photosynthetic reaction center in a ringlike manner. Isolated in a detergent solution, its appearance either as a ringlike complex (called B873 and absorbing at 873 nm), subunit dimer (called B820 and absorbing at 820 nm), or monomeric form (called B777 and absorbing at 777 nm) is strongly temperature-dependent. In thermodynamic equilibria between B820 and B873, intermediate-sized complexes have also been observed that have absorption maxima around 850 nm. It is unknown whether these structures appear as intermediates in the kinetic B820-B873 (dis)assembly reaction. In this paper disassembly of the light-harvesting complex into its dimeric subunits was followed spectroscopically on a time scale up to 200 ms, upon applying an infrared laser-induced temperature jump. The full dissociation process appears to take place on a time scale of tens to hundreds of milliseconds, the rates becoming faster at higher starting temperatures. Applying the same technique, the dissociation reaction of dimeric subunits into monomers also could be established. This dissociation process occurred on a much faster time scale and took place within the 500 micros response time of our detection system.
Protein folding is difficult to simulate with classical molecular dynamics. Secondary structure motifs such as alpha-helices and beta-hairpins can form in 0.1-10 micros (ref. 1), whereas small proteins have been shown to fold completely in tens of microseconds. The longest folding simulation to date is a single 1- micro s simulation of the villin headpiece; however, such single runs may miss many features of the folding process as it is a heterogeneous reaction involving an ensemble of transition states. Here, we have used a distributed computing implementation to produce tens of thousands of 5-20-ns trajectories (700 micros) to simulate mutants of the designed mini-protein BBA5. The fast relaxation dynamics these predict were compared with the results of laser temperature-jump experiments. Our computational predictions are in excellent agreement with the experimentally determined mean folding times and equilibrium constants. The rapid folding of BBA5 is due to the swift formation of secondary structure. The convergence of experimentally and computationally accessible timescales will allow the comparison of absolute quantities characterizing in vitro and in silico (computed) protein folding.
Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.
Quantitative models and experiments are revealing how the folding free energy surface of a protein is sculpted by sequence and environment. The sometimes conflicting demands of folding, structure and function determine which folding pathways, if any, dominate. Recent advances include experimental estimates of diffusive barrier-crossing times, the observation of ultrafast folders amenable to full-atom simulation, the use of thermodynamic tuning and nonconservative mutations to probe 'hidden' parts of the free energy surface, and a complete microscopic theory of folding.