Szabolcs Osváth

Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3‐phosphoglycerate kinase

3‐Phosphoglycerate kinase (PGK) is a typical two‐domain hinge‐bending enzyme with a well‐structured interdomain region. The mechanism of domain–domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N‐ or in the C‐terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates: 3‐phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain–domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well‐defined backbone and side‐chain H bonds, and hydrophobic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain–domain communication. Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission of the substrate‐induced conformational effects from one domain to the other. The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented [Sinev MA, Razgulyaev OI, Vas M, Timchenko AA & Ptitsyn OB (1989) Eur J Biochem180, 61–66]. Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates.

Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases

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.

Hierarchic Finite Level Energy Landscape Model TO DESCRIBE THE REFOLDING KINETICS OF PHOSPHOGLYCERATE KINASE

One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we provide the strongest evidence so far of such hierarchy and determine the time constants and weights of the kinetic components of the suggested hierarchic energy landscape. To our knowledge, the idea of hierarchical folding energy barriers has never been tested over such a broad timescale. Refolding of yeast phosphoglycerate kinase was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 ms to 15 min. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no a priori assumptions about the folding landscape. The result was a hierarchic finite level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase from 1 ms to 15 min. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step.

The structure of horseradish peroxidase C characterized as a molten globule state after Ca2+ depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca(2+) ions. Earlier studies suggested a critical role of one of the bound Ca(2+) ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca(2+)-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca(2+)-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca(2+), perturbs a H-bonding network connecting this Ca(2+) to the heme crevice that involves the distal water. The measured effects of Ca(2)(+) depletion can be interpreted as supporting a structural role for the distal Ca(2+) and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.

Thermodynamics and Kinetics of the Pressure Unfolding of Phosphoglycerate Kinase

Due to the relationship between compressibility and volume fluctuations, high-pressure studies provide vital insight into protein dynamics and function. Most high-pressure experiments were performed on small and fast folding proteins or model peptides. Here we show that a detailed kinetic study is necessary to extract reliable information from the high-pressure-induced structural conversion of large, slowly folding proteins. The pressure-jump unfolding kinetics of yeast phosphoglycerate kinase was recorded at pressures between 50 and 150 MPa. The time dependence of the conformational state of the protein was followed by tryptophan fluorescence measurements from 30 s to 2 h. The observed changes were described by a three-state model, and the volume change and the activation volume as well as the midpoint pressure of the transitions between the folded, intermediate, and unfolded states were determined. An interesting feature of the pressure unfolding of phosphoglycerate kinase was that the unfolding process speeds up with increasing pressure, which is the consequence of negative activation volumes for the folded --> intermediate, intermediate --> unfolded, and unfolded --> intermediate transitions.

Comparing the folding and misfolding energy landscapes of phosphoglycerate kinase.

Partitioning of polypeptides between protein folding and amyloid formation is of outstanding pathophysiological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state, and monitored by fluorescence from 10 ms to 20 days. Solvent conditions were gradually shifted between folding and amyloidogenesis, and the properties of the energy landscape governing structure formation were reconstructed. A gradual transition of the energy landscape between folding and amyloid formation was observed. In the early steps of both folding and misfolding, the protein searches through a hierarchically structured energy landscape to form a molten globule in a few seconds. Depending on the conditions, this intermediate either folds to the native state in a few minutes, or forms amyloid fibers in several days. As conditions are changed from folding to misfolding, the barrier separating the molten globule and native states increases, although the barrier to the amyloid does not change. In the meantime, the native state also becomes more unstable and the amyloid more stable. We conclude that the lower region of the energy landscape determines the final protein structure.

Domain interactions direct misfolding and amyloid formation of yeast phosphoglycerate kinase

There are proteins that are built of two structural domains and are deposited full‐length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single‐ and multidomain proteins, no published studies can be found that address the role of the domain–domain interactions during misfolding and amyloid formation. By the discovery of the role of domain–domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild‐type yeast phosphoglycerate kinase and single‐tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild‐type protein. Misfolding and amyloid formation was followed in stopped‐flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain–domain interactions direct the misfolding and amyloid formation mechanism. Proteins 2006.

Motion Based X-Ray Imaging Modality

A new X-ray imaging method (patent pending) was developed to visualize function-related motion information. We modify existing X-ray imaging methods to provide four images without increasing the necessary measurement time or radiation dose. The most important of these images is a new “kinetic” image that represents motions inside the object or living body. The motion-based contrast of the kinetic image can help visualize details that were not accessible before. The broad range of the movements and high sensitivity of the method are illustrated by imaging the mechanics of a working clock and the chest of a living African clawed frog (Xenopus laevis). The heart, valves, aorta, and lungs of the frog are clearly visualized in spite of the low soft tissue contrast of the animal. The new technology also reconstructs a “static” image similar to the existing conventional X-ray image. The static image shows practically the same information as the conventional image. The new technology presents two more images which show the point-wise errors of the static and kinetic images. This technique gives a better estimation of errors than present methods because it is based entirely on measured data. The new technology could be used in imaging cardiopulmonary movements, nondestructive testing, or port security screening.

Recovery of functional enzyme from amyloid fibrils

Amyloid deposits, which accumulate in numerous diseases, are the final stage of multi‐step protein conformational‐conversion and oligomerization processes. The underlying molecular mechanisms are not fully understood, and particularly little is known about the reverse reaction. Here we show that phosphoglycerate kinase amyloid fibrils can be converted back into native protein. We achieved recovery with 60% efficiency, which is comparable to the success rate of the unfolding‐refolding studies, and the recovered enzyme was folded, stable and fully active. The key intermediate stages in the recovery process are fibril disassembly and unfolding followed by spontaneous protein folding.

Role of Domain Interactions in the Collective Motion of Phosphoglycerate Kinase

Protein function is governed by the underlying conformational dynamics of the molecule. The experimental and theoretical work leading to contemporary understanding of enzyme dynamics was mostly restricted to the large-scale movements of single-domain proteins. Collective movements resulting from a regulatory interplay between protein domains is often crucial for enzymatic activity. It is not clear, however, how our knowledge could be extended to describe collective near-equilibrium motions of multidomain enzymes. We examined the effect of domain interactions on the low temperature near equilibrium dynamics of the native state, using phosphoglycerate kinase as model protein. We measured thermal activation of tryptophan phosphorescence quenching to explore millisecond-range protein motions. The two protein domains of phosphoglycerate kinase correspond to two dynamic units, but interdomain interactions link the motion of the two domains. The effect of the interdomain interactions on the activation of motions in the individual domains is asymmetric. As the temperature of the frozen protein is increased from the cryogenic, motions of the N domain are activated first. This is a partial activation, however, and the full dynamics of the domain becomes activated only after the activation of the C domain.