No abstract

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.

A minimal kinetic model of the photocycle, including both quinone (Q-6) reduction at the secondary quinone-binding site and (mammalian) cytochrome c oxidation at the cytochrome docking site of isolated reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides, was elaborated and tested by cytochrome photooxidation under strong continuous illumination. The typical rate of photochemical excitation by a laser diode at 810 nm was 2.200 s-1, and the rates of stationary turnover of the reaction center (one-half of that of cytochrome photooxidation) were 600 +/- 70 s-1 at pH 6 and 400 +/- 50 s-1 at pH 8. The rate of turnover showed strong pH dependence, indicating the contribution of different rate-limiting processes. The kinetic limitation of the photocycle was attributed to the turnover of the cytochrome c binding site (pH < 6), light intensity and quinone/quinol exchange (6 < pH < 8), and proton-coupled second electron transfer in the quinone acceptor complex (pH > 8). The analysis of the double-reciprocal plot of the rate of turnover versus light intensity has proved useful in determining the light-independent (maximum) turnover rate of the reaction center (445 +/- 50 s-1 at pH 7.8).

The experimental and theoretical studies that led to our present understanding of protein structural changes and their role in enzyme function were mostly carried out on small single-domain proteins [1]. Most enzymes, however, are built of several domains. The mechanism of transmission of molecular interactions over large distances (e.g. from one domain to the other) within the molecule, the role of substrates in 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

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.

The aim of this work is to shed more light on the effect of domain-domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain-domain interactions direct the folding of both domains, but in an asymmetric way.

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.

 Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.