Derek R. Dee

Direct observation of transition paths during the folding of proteins and nucleic acids.

How biomolecules fold In order to fold, biomolecules must search a conformational energy landscape to find low-energy states. There are peaks in the landscape where the molecules must occupy unstable conformations for a short time. Neupane et al. used optical tweezers to observe these transition paths directly for single nucleic acid and protein molecules (see the Perspective by Wolynes). They measured a distribution of times taken to cross the transition path and found that the shape of the distribution agrees well with theory that assumes one-dimensional diffusion over the landscape. Science, this issue p. 239; see also p. 150 Optical tweezers reveal how a protein and a DNA hairpin cross the barrier between the folded and unfolded states. [Also see Perspective by Wolynes] Transition paths, the fleeting trajectories through the transition states that dominate the dynamics of biomolecular folding reactions, encapsulate the critical information about how structure forms. Owing to their brief duration, however, they have not previously been observed directly. We measured transition paths for both nucleic acid and protein folding, using optical tweezers to observe the microscopic diffusive motion of single molecules traversing energy barriers. The average transit times and the shapes of the transit-time distributions agreed well with theoretical expectations for motion over the one-dimensional energy landscapes reconstructed for the same molecules, validating the physical theory of folding reactions. These measurements provide a first look at the critical microscopic events that occur during folding, opening exciting new avenues for investigating folding phenomena.

Protein misfolding occurs by slow diffusion across multiple barriers in a rough energy landscape

Significance Structural transitions in proteins are characterized by the coefficient for intrachain diffusion, D, which determines the transition kinetics and reveals microscopic properties of the interactions governing folding. D has been measured for unfolded proteins and for native folding, but never for misfolding and aggregation, despite the importance of kinetics for driving these processes. We used single-molecule force spectroscopy to observe the misfolding of individual prion protein (PrP) molecules into stable, nonnative dimers. By reconstructing the energy landscape for dimer misfolding, we compared D for misfolding of PrP to that for native folding. Diffusion was 1,000-fold slower for misfolding, reflecting significant additional roughness in the energy landscape and confirming quantitatively the long-held hypothesis that misfolding landscapes are rougher than native landscapes. The timescale for the microscopic dynamics of proteins during conformational transitions is set by the intrachain diffusion coefficient, D. Despite the central role of protein misfolding and aggregation in many diseases, it has proven challenging to measure D for these processes because of their heterogeneity. We used single-molecule force spectroscopy to overcome these challenges and determine D for misfolding of the prion protein PrP. Observing directly the misfolding of individual dimers into minimal aggregates, we reconstructed the energy landscape governing nonnative structure formation. Remarkably, rather than displaying multiple pathways, as typically expected for aggregation, PrP dimers were funneled into a thermodynamically stable misfolded state along a single pathway containing several intermediates, one of which blocked native folding. Using Kramers’ rate theory, D was found to be 1,000-fold slower for misfolding than for native folding, reflecting local roughening of the misfolding landscape, likely due to increased internal friction. The slow diffusion also led to much longer transit times for barrier crossing, allowing transition paths to be observed directly for the first time to our knowledge. These results open a new window onto the microscopic mechanisms governing protein misfolding.

Multifunctional aspartic peptidase prosegments

The structure-function relationships of aspartic peptidases (APs) (EC 3.4.23.X) have been extensively investigated, yet much remains to be elucidated regarding the various molecular mechanisms of these enzymes. Over the past years, APs have received considerable interest for food applications (e.g. cheese, fermented foods) and as potential targets for pharmaceutical intervention in human diseases including hypertension, cancer, Alzheimers disease, AIDS (acquired immune deficiency syndrome), and malaria. A deeper understanding of the structure and function of APs, therefore, will have a direct impact on the design of peptidase inhibitors developed to treat such diseases. Most APs are synthesized as zymogens which contain an N-terminal prosegment (PS) domain that is removed at acidic pH by proteolytic cleavage resulting in the active enzyme. While the nature of the AP PS function is not entirely understood, the PS can be important in processes such as the initiation of correct folding, protein stability, blockage of the active site, pH-dependence of activation, and intracellular sorting of the zymogen. This review summarizes the current knowledge of AP PS function (especially within the A1 family), with particular emphasis on protein folding, cellular sorting, and inhibition.

Structure-function characterization of the recombinant aspartic proteinase A1 from arabidopsis thaliana.

Aspartic proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric aspartic proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher alpha-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3-8) with the highest stability at pH 5-6, where 70-80% of the activity was retained after 1 month at 37 degrees C. Using calorimetry, a melting point of 79.6 degrees C was observed at pH 5.3. Cleavage profiles of insulin beta-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu(15)-Tyr(16) peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.

The prosegment catalyzes pepsin folding to a kinetically trapped native state.

Investigations of irreversible protein unfolding often assume that alterations to the unfolded state, rather than the nature of the native state itself, are the cause of the irreversibility. However, the present study describes a less common explanation for the irreversible denaturation of pepsin, a zymogen-derived aspartic peptidase. The presence of a large folding barrier combined with the thermodynamically metastable nature of the native state, the formation of which depends on a separate prosegment (PS) domain, is the source of the irreversibility. Pepsin is unable to refold to the native state upon return from denaturing conditions due to a large folding barrier (24.6 kcal/mol) and instead forms a thermodynamically stable, yet inactive, refolded state. The native state is kinetically stabilized by an unfolding activation energy of 24.5 kcal/mol, comparable to the folding barrier, indicating that native pepsin exists as a thermodynamically metastable state. However, in the presence of the PS, the native state becomes thermodynamically stable, and the PS catalyzes pepsin folding by stabilizing the folding transition state by 14.7 kcal/mol. Once folded, the PS is removed, and the native conformation exists as a kinetically trapped state. Thus, while PS-guided folding is thermodynamically driven, without the PS the pepsin energy landscape is dominated by kinetic barriers rather than by free energy differences between native and denatured states. As pepsin is the archetype of a broad class of aspartic peptidases of similar structure and function, and many require their PS for correct folding, these results suggest that the occurrence of native states optimized for kinetic rather than thermodynamic stability may be a common feature of protein design.

Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90-232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al(3+)-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al(3+), Zn(2+), and free-base). Buffer ionic strength also affected the binding, with K(d) increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4-5 PcTS molecules per PrP molecule.

Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin

Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, Ki, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min(-1), five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.

Single-molecule approaches to prion protein misfolding

The structural conversion of the prion protein PrP into a transmissible, misfolded form is the central element of prion disease, yet there is little consensus as to how it occurs. Key aspects of conversion into the diseased state remain unsettled, from details about the earliest stages of misfolding such as the involvement of partially- or fully-unfolded intermediates to the structure of the infectious state. Part of the difficulty in understanding the structural conversion arises from the complexity of the underlying energy landscapes. Single molecule methods provide a powerful tool for probing complex folding pathways as in prion misfolding, because they allow rare and transient events to be observed directly. We discuss recent work applying single-molecule probes to study misfolding in prion proteins, and what it has revealed about the folding dynamics of PrP that may underlie its unique behavior. We also discuss single-molecule studies probing the interactions that stabilize non-native structures within aggregates, pointing the way to future work that may help identify the microscopic events triggering pathogenic conversion. Although single-molecule approaches to misfolding are relatively young, they have a promising future in prion science.

Dynamics of Thermodynamically Stable, Kinetically Trapped, and Inhibitor-Bound States of Pepsin

The pepsin folding mechanism involves a prosegment (PS) domain that catalyzes folding, which is then removed, resulting in a kinetically trapped native state. Although native pepsin (Np) is kinetically stable, it is irreversibly denatured due to a large folding barrier, and in the absence of the PS it folds to a more thermodynamically stable denatured state, termed refolded pepsin (Rp). This system serves as a model to understand the nature of kinetic barriers and folding transitions between compact states. Quasielastic neutron scattering (QENS) was used to characterize and compare the flexibility of Np, as a kinetically trapped state, with that of Rp, as a thermodynamically stable fold. Additionally, the dynamics of Np were compared with those of a partially unfolded form and a thermally stabilized, inhibitor-bound form. QENS revealed length-scale-dependent differences between Np and Rp on a picosecond timescale and indicated greater flexibility in Np, leading to the conclusion that kinetic stabilization likely does not correspond to reduced internal dynamics. Furthermore, large differences were observed upon inhibition, indicating that QENS of proteins in solution may prove useful for examining the role of conformational entropy changes in ligand binding.

The native conformation of plasmepsin II is kinetically trapped at neutral pH

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5-8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37 °C for 2h, followed by adjustment to a pH in the range of 6.5-8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25 °C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5 kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.