Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α₃D using atomic force microscopy (AFM)–based force spectroscopy. α₃D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α₃D’s configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy’s ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α₃D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam–modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 k_BT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Single-molecule force spectroscopy (SMFS) has been remarkably successful across broad classes of biological molecules (RNA, DNA, and proteins) (1–5). A particularly fruitful data acquisition regime probes multiple back-and-forth folding/unfolding transitions at near-equilibrium and equilibrium conditions (6–9). This methodology efficiently yields numerous transitions and therefore a wealth of kinetic data, one-dimensional (1D) free-energy landscape parameters, and even a full 1D projection of the free-energy landscape along the stretching axis (10, 11). The standard SMFS assay has the molecule of interest tethered via a flexible linker to the force probe, such as an optically trapped bead or an atomic force microscopy (AFM) cantilever (Fig. 1A). These micrometer-sized force probes are the primary measurement (x_meas) but have finite response time and are therefore coupled to, but do not precisely track, molecular dynamics (x_prot) (Fig. 1B) (12–14). Additionally, the flexible linker’s compliance modifies this coupling between the molecule and the force probe. Linkers stretched at a finite force (F) can even create an entropic barrier not present in the absence of applied force (15, 16). More generally, there is an expanding set of theoretical and experimental studies (12–30) investigating how such instrumental and assay parameters affect the underlying biomolecular dynamics and whether the measured dynamics are dominated by the instrument used to measure them.Open in a separate windowFig. 1.Probing the folding and unfolding dynamics of a globular protein by SMFS. (A) Cartoon showing a polyprotein consisting of a single copy of α₃D (blue) and two copies of NuG2 (red) stretched with an atomic force microscope. At low forces, the mechanically labile α₃D repeatedly unfolds and refolds as detected by a change in cantilever deflection. (B) A conceptual two-dimensional free-energy landscape shows the underlying protein extension (x_prot) and the experimentally measured extension (x_meas). The macroscopic force probe has finite temporal resolution, and the application of force can introduce an entropic barrier between resolved states. (C) The sum of the equilibrium folding and unfolding rates for α₃D in a strong denaturant (5 to 6 M urea) as a function of pH as determined in a prior smFRET study (37). (D) A conceptual sketch of α₃D’s 1D free-energy landscape deduced by a combination of smFRET and molecular dynamics studies based on Ref. 37. The dramatic increase in α₃D’s kinetics at low pH shown in panel C was explained as increased configurational diffusion along a smooth rather than a rough energy landscape.AFM characterization of proteins is widely used (1–5) and therefore is an important experimental regime to explore, distinct from numerous studies investigating instrumental effects on nucleic acid hairpins measured with optical traps (17, 18, 23, 24, 26, 31). Historically, limited force precision and stability coupled with the slow response of the force probe has made it challenging to perform AFM-based equilibrium and near-equilibrium studies (32) and thereby difficult to quantify the role of instrumental artifacts. Recent work using a standard gold-coated cantilever concluded that the equilibrium dynamics of the fast-folding protein gpW were dominated by the dynamics of the cantilever diffusing on a force-induced entropic barrier (29). Such results raise significant concerns about interpreting rates or landscapes measured in AFM studies of globular protein folding and thereby motivate the following question: How do variations in intrinsic protein folding dynamics manifest in AFM-based studies, particularly in an experimental regime dominated by an instrument-induced entropic barrier?Here, we address this question by directly modulating a globular protein’s underlying folding dynamics without significantly changing the height of the barrier or the free-energy difference between the states. To do so, we studied α₃D using AFM-based force spectroscopy (Fig. 1A). The dynamics and energetics of α₃D, a computationally designed, fast-folding three-helix bundle of 73 amino acids (33, 34), have been studied by traditional ensemble (33) and single-molecule fluorescence resonance energy transfer (smFRET) (35–39) assays. Equilibrium smFRET studies in chemical denaturants showed accelerated folding/unfolding kinetics as pH was reduced (35). A subsequent landmark paper (37) combined state-of-the-art smFRET and microsecond-long, all-atom molecular dynamics simulations to show that this acceleration resulted from suppression of nonnative contacts changing the local roughness of the 1D landscape (Fig. 1 C and D) rather than a change in the height or the overall shape of the barrier between states. In the context of Kramers theory (40), this roughness manifests as a change in D, the conformational diffusion coefficient along the 1D landscape. The authors concluded that most, if not all, of the 14-fold change in folding kinetics came from an increase in D. This pH-dependent change in kinetics serves as a benchmark of α₃D’s dynamics in the absence of the force probe and associated linker. In other words, we will leverage conditions known to modulate the rate of folding along the molecular coordinate (x_prot) while measuring the consequence of that change on the measured coordinate (x_meas) (Fig. 1B).While α₃D provides a conceptually attractive means to modulate intrinsic molecular dynamics, it presents significant experimental challenges. Like gpW (28, 29), α₃D unfolds at a low force (< 15 pN) by AFM standards (2, 3, 41) and exhibits even faster folding kinetics under force. Thus, spatiotemporal resolution is critical, and instrumentation limitations are expected to be even more pronounced. Force drift is also a critical issue, particularly for extended assays (>1 to 100 s) because standard gold-coated cantilevers exhibit significant force drift (42); yet, equilibrium assays of structured RNA (6) and proteins (9) are sensitive to sub-pN changes in F. We therefore used focused ion beam (FIB)–modified cantilevers (32, 43) that combine sub-pN stability over 100 s (43, 44) with a ∼13-fold improvement in spatiotemporal precision compared with the standard cantilever used in the aforementioned AFM study characterizing gpW (29). This stability in conjunction with a newly designed polyprotein construct allowed us to measure an individual α₃D unfold and fold over 5,000 times and for periods up to 1 h using both constant velocity (v) and equilibrium (v = 0) data acquisition protocols. Rates derived from both the equilibrium and dynamic data recapitulated α₃D’s pH-dependent kinetics from smFRET. However, the reconstructed 1D folding-energy landscape was consistent with the predicted entropic barrier and therefore encodes no information about α₃D’s folding landscape beyond ΔG₀, the thermodynamic stability of α₃D. Importantly, rate analysis yielded the expected asymmetric distance to the transition state from the folded and unfolded state and revealed a significant increase in the distance to the unfolding transition state as pH was lowered. These studies demonstrate that AFM-force spectroscopy can track changes in intrinsic protein dynamics with high precision, even in mechanically labile, fast-folding systems.     

Structural and kinetic mapping of side-chain exposure onto the protein energy landscape

Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated.     

Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: evidence for a kinetic partitioning mechanism

Kinetic partitioning is predicted to be a general mechanism for proteins to fold into their well defined native three-dimensional structure from unfolded states following multiple folding pathways. However, experimental evidence supporting this mechanism is still limited. By using single-molecule atomic force microscopy, here we report experimental evidence supporting the kinetic partitioning mechanism for mechanical unfolding of T4 lysozyme, a small protein composed of two subdomains. We observed that on stretching from its N and C termini, T4 lysozyme unfolds by multiple distinct unfolding pathways: the majority of T4 lysozymes unfold in an all-or-none fashion by overcoming a dominant unfolding kinetic barrier; and a small fraction of T4 lysozymes unfold in three-state fashion involving unfolding intermediate states. The three-state unfolding pathways do not follow well defined routes, instead they display variability and diversity in individual unfolding pathways. The unfolding intermediate states are local energy minima along the mechanical unfolding pathways and are likely to result from the residual structures present in the two subdomains after crossing the main unfolding barrier. These results provide direct evidence for the kinetic partitioning of the mechanical unfolding pathways of T4 lysozyme, and the complex unfolding behaviors reflect the stochastic nature of kinetic barrier rupture in mechanical unfolding processes. Our results demonstrate that single-molecule atomic force microscopy is an ideal tool to investigate the folding/unfolding dynamics of complex multimodule proteins that are otherwise difficult to study using traditional methods.     

Protein folding and unfolding on a complex energy landscape

Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa. Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism. However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.     

Multiple pathways of angiotensin production in the blood vessel wall: evidence, possibilities and hypotheses

In summary, multiple pathways of angiotensin production may exist in the blood vessel wall (Fig. 1), in addition to the well described renin--ACE enzymatic axis. It is not known whether these enzymatic pathways represent in vitro phenomena, or authentic in vivo alternate pathways which are activated only when the renin--ACE pathway is blocked, or whether they are operative at all times in the vessel wall. If these multiple pathways are functional, then the vascular angiotensin system may be very complicated, and may vary in different pathophysiological states. Future research in this area is likely to yield important and novel information on the in vivo pathways of production and function of vascular angiotensin.     

Multiple routes lead to the native state in the energy landscape of the beta-trefoil family

In general, the energy landscapes of real proteins are sufficiently well designed that the depths of local energetic minima are small compared with the global bias of the native state. Because of the funneled nature of energy landscapes, models that lack energetic frustration have been able to capture the main structural features of the transition states and intermediates found in experimental studies of both small and large proteins. In this study we ask: Are the experimental differences in folding mechanisms among members of a particular structural family due to local topological constraints that deviate from the tertiary fold common to the family? The beta-trefoil structural family members IL-1beta, hisactophilin, and acidic/basic FGFs were chosen to address this question. It has been observed that the topological landscape of the beta-trefoils allows for the population of diverse, geometrically disconnected routes that provide energetically similar but structurally distinct ways for this family to fold. Small changes in topology or energetics can alter the preferred route. Taken together, these results indicate that the global fold of the beta-trefoil family determines the energy landscape but that the routes accessed on that landscape might differ as a result of functional requirements of the individual family members.     

Myosin V processivity: multiple kinetic pathways for head-to-head coordination

Myosin V, a double-headed molecular motor, transports organelles within cells by walking processively along actin, a process that requires coordination between the heads. To understand the mechanism underlying this coordination, processive runs of single myosin V molecules were perturbed by varying nucleotide content. Contrary to current views, our results show that the two heads of a myosin V molecule communicate, not through any one mechanism but through an elaborate system of cooperative mechanisms involving multiple kinetic pathways. These mechanisms introduce redundancy and safeguards that ensure robust processivity under differing physiologic demands.     

Trp-cage: folding free energy landscape in explicit water

Trp-cage is a 20-residue miniprotein, which is believed to be the fastest folder known so far. In this study, the folding free energy landscape of Trp-cage has been explored in explicit solvent by using an OPLSAA force field with periodic boundary condition. A highly parallel replica exchange molecular dynamics method is used for the conformation space sampling, with the help of a recently developed efficient molecular dynamics algorithm P3ME/RESPA (particle-particle particle-mesh Ewald/reference system propagator algorithm). A two-step folding mechanism is proposed that involves an intermediate state where two correctly formed partial hydrophobic cores are separated by an essential salt-bridge between residues Asp-9 and Arg-16 near the center of the peptide. This metastable intermediate state provides an explanation for the superfast folding process. The free energy landscape is found to be rugged at low temperatures, and then becomes smooth and funnel-like above 340 K. The lowest free energy structure at 300 K is only 1.50 A Calpha-RMSD (Calpha-rms deviation) from the NMR structures. The simulated nuclear Overhauser effect pair distances are in excellent agreement with the raw NMR data. The temperature dependence of the Trp-cage population, however, is found to be significantly different from experiment, with a much higher melting transition temperature above 400 K (experimental 315 K), indicating that the current force fields, parameterized at room temperature, need to be improved to correctly predict the temperature dependence.     

Direct observation of tiers in the energy landscape of a chromoprotein: a single-molecule study

Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed.     

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen–deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

Ubiquitin is an 8.5-kDa protein appended to target proteins as a posttranslational modification (PTM). Typically, ubiquitin is conjugated to the primary amine of substrate lysine residues, though noncanonical linkages to serine and cysteine also exist in vivo. Ubiquitin itself contains seven lysine residues, which allows building of ubiquitin chains with various linkages and topologies. Ubiquitination is most typically associated with targeting condemned proteins to the 26S proteasome for degradation; however, it is also involved in a large and ever-growing list of crucial regulatory, nondegradative cellular processes (1). A complex and highly regulated enzymatic cascade attaches ubiquitin to substrates and therefore plays a key role in determining the specific downstream effects of an individual ubiquitination event. There are several hundred E3 ligases, the terminal enzymes in this cascade (2), which give rise to broad proteome coverage and allow for some level of site specificity (3, 4).Multiple different ubiquitin chain linkages and topologies bind with high affinity to proteasomal ubiquitin receptors and promote degradation (5–8). However, the presence of a ubiquitin tag alone is not sufficient to ensure proteasomal degradation. In fact, a substantial proportion of ubiquitin-modified proteins that interact with the 26S proteasome are ultimately released (9, 10) and not degraded. The proteasome also relies on substrate conformational properties, initiating degradation at an unstructured region on the condemned protein (11, 12). Much work has been done to understand the requirements of this unstructured region with regard to length, sequence composition, and topological position (13–15), yet at least 30% of known proteasome clients lack such a region (16). While evidence suggests that well-folded proteins are processed by diverse cellular unfoldases, such as Cdc48/p97/VCP (17, 18), an intriguing possibility is that the ubiquitin modification itself can modulate the conformational landscape and thus regulate proteasome substrate selection. Simulations have suggested that ubiquitination can destabilize the folded state of the substrate protein, thereby allowing it to more readily adopt unfolded or partially unfolded conformations (19, 20).Recently, we demonstrated that this is indeed the case: ubiquitin can exert significant effects on a substrate’s energy landscape depending on the site of ubiquitination and the identity of the substrate protein. Moreover, these changes can have direct consequences for proteasomal processing (21). By examining the energetic effects of native, isopeptide-linked ubiquitin attachment to three different sites within the small protein barstar from Bacillus amyloliquefaciens, we found that ubiquitin attached at either lysine 2 or lysine 60 destabilizes the protein both globally and via subglobal fluctuations, and we thus refer to these residues as sensitive sites. By contrast, ubiquitination at lysine 78 produces little effect on the energy landscape (21), and we therefore term it a nonsensitive site. Another study found that ubiquitin, appended through a nonnative linkage, can destabilize a folded substrate as measured by changes in the midpoints for thermally induced unfolding transitions (22).Ubiquitination at the two sensitive sites in barstar increases the population of partially unfolded, high-energy states on the landscape sufficient for proteasomal engagement and degradation. Ubiquitination at the single nondestabilizing site does not allow for proteasomal degradation. These results suggest that ubiquitin-mediated destabilization can reveal an obligate unstructured region in substrates that otherwise lack such a region. Furthermore, ubiquitination at sensitive sites results in more rapid degradation of these barstar variants when a proteasome-engageable unstructured tail is fused to their C termini (21).This previous work clearly demonstrates that ubiquitin-mediated changes to the protein landscape can play an important role in proteasomal selectivity and processing; it did not, however, uncover the molecular mechanisms through which these site-specific effects arise. Here, we interrogate the molecular mechanisms of ubiquitin-induced changes for these same single-lysine variants of barstar. We investigate differences in the intrinsic dynamics of these regions within barstar and differences in how the protein responds to ubiquitination at these individual sites. We employed two sets of complementary approaches: 1) NMR and HDX-MS (hydrogen–deuterium exchange mass spectrometry) to characterize the equilibrium conformational fluctuations of the substrate protein in the presence and absence of ubiquitin and 2) molecular dynamics (MD) simulations to track the position of every atom in barstar, in the presence and absence of ubiquitin, starting from its native conformation over the timescale of microseconds.We find that ubiquitination has only subtle effects on the native structure of barstar. Ubiquitination at the sensitive sites, however, selectively increases fluctuations that expose barstar’s C terminus. While both of the sensitive sites arise in regions of barstar with relatively high conformational flexibility, the observed destabilization appears to occur through different thermodynamic mechanisms. By contrast, ubiquitination at the nonsensitive site has a protective effect on barstar’s C terminus. Thus, the effects of ubiquitination at each site are highly dependent on the local context. This mechanistic understanding of the site-specific effects of ubiquitination should aid in developing predictive models of the energetic consequences of individual ubiquitination events and also of the ways in which aberrant lysine targeting leads to disease (23–25).     

Protein folding pathways from replica exchange simulations and a kinetic network model

We present an approach to the study of protein folding that uses the combined power of replica exchange simulations and a network model for the kinetics. We carry out replica exchange simulations to generate a large ( approximately 10(6)) set of states with an all-atom effective potential function and construct a kinetic model for folding, using an ansatz that allows kinetic transitions between states based on structural similarity. We use this network to perform random walks in the state space and examine the overall network structure. Results are presented for the C-terminal peptide from the B1 domain of protein G. The kinetics is two-state after small temperature perturbations. However, the coil-to-hairpin folding is dominated by pathways that visit metastable helical conformations. We propose possible mechanisms for the alpha-helix/beta-hairpin interconversion.     

Folding domain B of protein A on a dynamically partitioned free energy landscape

The B domain of staphylococcal protein A (BdpA) is a small helical protein that has been studied intensively in kinetics experiments and detailed computer simulations that include explicit water. The simulations indicate that BdpA needs to reorganize in crossing the transition barrier to facilitate folding its C-terminal helix (H3) onto the nucleus formed from helices H1 and H2. This process suggests frustration between two partially ordered forms of the protein, but recent φ value measurements indicate that the transition structure is relatively constant over a broad range of temperatures. Here we develop a simplistic model to investigate the folding transition in which properties of the free energy landscape can be quantitatively compared with experimental data. The model is a continuation of the Muñoz–Eaton model to include the intermittency of contacts between structured parts of the protein, and the results compare variations in the landscape with denaturant and temperature to φ value measurements and chevron plots of the kinetic rates. The topography of the model landscape (in particular, the feature of frustration) is consistent with detailed simulations even though variations in the φ values are close to measured values. The transition barrier is smaller than indicated by the chevron data, but it agrees in order of magnitude with a similar α-carbon type of model. Discrepancies with the chevron plots are investigated from the point of view of solvent effects, and an approach is suggested to account for solvent participation in the model.     

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA-. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA- was associated with similar time constants of 1.5 ps and 1. 7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA- and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.     

Erythropoietin production: evidence for multiple oxygen sensing pathways

 The production of erythropoietin (Epo), the glycoprotein hormone which controls red blood cell formation, is regulated by feedback mechanisms sensing tissue oxygenation. The mechanism of the putative oxygen sensor has yet to be elucidated. There is evidence that at least two pathways participate in hypoxia signal transduction. One appears to involve a specific haem protein, and a second implicates reactive oxygen species (ROS). Iron catalyses the generation of intracellular ROS and therefore alters the cellular redox state. We have investigated the effect of modulating intracellular iron content on Epo production in Hep 3B cells. Iron chelation stimulates Epo production at 20% O₂ and enhances Epo production at 1% O₂, but it has no additive effect on cobalt-induced Epo production. Excess molar iron inhibited Epo production in response to hypoxia, desferrioxamine (DFO) and cobalt chloride and inhibited the DFO-enhancing effect of hypoxia-induced Epo production. We found that sulphydryl oxidising agents exert a differential inhibitory effect on hypoxia-induced versus DFO-induced Epo production, providing further evidence that multiple pathways of oxygen sensing exist. Received: September 21, 1998 / Accepted: March 15, 1999     

Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments

Recent theoretical and experimental results point to the existence of small barriers to protein folding. These barriers can even be absent altogether, resulting in a continuous folding transition (i.e., downhill folding). With small barriers, the detailed properties of folding ensembles may become accessible to equilibrium experiments. However, further progress is hampered because folding experiments are interpreted with chemical models (e.g., the two-state model), which assume the existence of well defined macrostates separated by arbitrarily high barriers. Here we introduce a phenomenological model based on the classical Landau theory for critical transitions. In this physical model the height of the thermodynamic free energy barrier and the general properties of the folding ensemble are directly obtained from the experimental data. From the analysis of differential scanning calorimetry data alone, our model identifies the presence of a significant (>35 kJ/mol) barrier for the two-state protein thioredoxin and the absence of a barrier for BBL, a previously characterized downhill folding protein. These results illustrate the potential of our approach for extracting the general features of protein ensembles from equilibrium folding experiments.     

Human myoglobin recognition of oxygen: dynamics of the energy landscape

Femtosecond to nanosecond dynamics of O(2) rebinding to human WT myoglobin and its mutants, V68F and I107F, have been studied by using transient absorption. The results are compared with NO rebinding. Even though the immediate environment around the heme binding site is changed by the mutations, the picosecond geminate rebinding of oxygen is at most minimally affected. On the other hand, the V68F (E11) mutation causes drastic differences in rebinding on the nanosecond time scale, whereas the effect of the I107F (G8) mutation remains relatively small within our 10-ns time window. Unlike traditional homogeneous kinetics and molecular dynamics collisional simulations, we propose a "bifurcation model" for populations of directed and undirected dynamics on the ultrafast time scale, reflecting the distribution of initial protein conformations. The major mutation effect occurs on the time scale on which global protein conformational change is possible, consistent with transitions between the conformations of directed and undirected population playing a role in the O(2) binding. We discuss the relevance of these findings to the bimolecular function of the protein.     

Multiple signaling pathways converge on beta-catenin in thyroid cancer.

The beta-catenin pathway has been conclusively demonstrated to regulate differentiation and patterning in multiple model systems. In thyroid cancer, alterations are often seen in proteins that regulate beta-catenin, including those of the RAS, PI3K/AKT, and peroxisome proliferation activated receptor-gamma (PPARgamma) pathways, and evidence from the literature suggests that beta-catenin may play a direct role in the dedifferentiation commonly observed in late-stage disease. RET/PTC rearrangements are frequent in thyroid cancer and appear to be exclusive from mutational events in RAS and BRAF. Activation of AKT by phosphatidylinositide-3 kinase (PI3K), a RAS effector, results in GSK3beta phosphorylation and deactivation and subsequent beta-catenin upregulation in thyroid cancer. Activating mutations in beta-catenin, which have been demonstrated in late-stage thyroid tumors, correlate with beta-catenin nuclear localization and poor prognosis. We hypothesize that activation of the RAS, PI3K/AKT, and PPARgamma pathways ultimately impinges upon beta-catenin. We further propose that if mutations in BRAF, RAS, and RET/PTC rearrangements are mutually exclusive in certain thyroid tumors or tumor types, as has already been shown for papillary thyroid cancer, then these interconnected pathways may cooperate in the initiation and promotion of the disease. We believe that clinical benefit for thyroid cancer patients could be derived from disrupting the middle or distal pathway effectors of these pathways, such as AKT or beta-catenin.     

Experimental evidence for a kinetic and electrochemical memory in enzyme membranes.

The existence of hysteresis phenomena in artificial enzyme membranes due to the coupling of simple kinetic enzyme properties with diffusion transport processes is reported. The intramembrane pH of a urease coating on the surface of a glass pH electrode exhibits a hysteresis loop when the pH of the bulk solution varies cyclically. The steady-state potential of a urease membrane, as a function of the substrate concentration in the bulk solution, also exhibits a memory effect. The influence of the membrane's history on its overall behavior is visualized by electron microscopy. We interpret the results in terms of a coupling between the enzyme reactions and diffusion processes, without taking into account molecular effects.