Digital Variance Angiography in Selective Lower Limb Interventions.

PURPOSE: To evaluate the potential benefits of digital variance angiography (DVA) in selective lower limb angiography and to compare the performance of 2 DVA algorithms (conventional DVA1 and the recently developed DVA2) to that of digital subtraction angiography (DSA). MATERIALS AND METHODS: From November 2019 to May 2020, 112 iodinated contrast media (ICM) and 40 carbon dioxide (CO2) angiograms were collected from 15 and 13 peripheral artery disease patients, respectively. The DVA files were retrospectively generated from the same unsubtracted source file as DSA. The objectively calculated contrast-to-noise ratio (CNR) and the subjective visual image quality of DSA, DVA1, and DVA2 images were statistically compared using the Wilcoxon signed-rank test. The images were evaluated by 6 radiologists (R.P.T., S.V., A.M.K., S.S.A., O.E., and J.S.) from 2 centers using a 5-grade Likert scale. RESULTS: Both DVA algorithms produced similar increase (at least 2-fold) in CNR values (P < .001) and significantly higher image quality scores than DSA, independent of the contrast agent used. The overall scores with ICM were 3.61 ± 0.05 for DSA, 4.30 ± 0.04 for DVA1, and 4.33 ± 0.04 for DVA2 (each P < .001 vs DSA). The scores for CO2 were 3.10 ± 0.14 for DSA, 3.63 ± 0.13 for DVA1 (P < .001 vs DSA), and 3.38 ± 0.13 for DVA2 (P < .05 vs DSA). CONCLUSIONS: DVA provides higher CNR and significantly better image quality in selective lower limb interventions irrespective of the contrast agent used. Between DVA algorithms, DVA1 is preferred because of its identical or better image quality than DVA2. DVA can potentially help the interventional decision process and its quality reserve might allow dose management (radiation/ICM reduction) in the future.

Kinetic Imaging in Lower Extremity Arteriography: Comparison to Digital Subtraction Angiography.

Purpose To compare the image quality produced by kinetic imaging in x-ray angiography and the current reference standard digital subtraction angiography (DSA). Materials and Methods This prospective observational crossover study enrolled 42 patients undergoing lower limb x-ray angiography between February and June 2017 (mean age, 68.7 years; age range, 49-89 years; 32 men [mean age, 67.1 years; age range, 49-89 years] and 10 women [mean age, 75 years; age range, 57-85 years]). Signal-to-noise ratios (SNRs) of DSA and kinetic image pairs were compared. Visual quality comparisons were also performed by specialists who used an online questionnaire. Interrater agreement was characterized by percent agreement and Fleiss k. Results A total of 1902 regions of interest were carefully selected in 110 image pairs to calculate and compare the SNRs. Median SNR in raw kinetic images was 3.3-fold and 2.3-fold higher than raw and postprocessed DSA images, respectively. A total of 232 pairs of raw and postprocessed kinetic images were compared. It was indicated that postprocessing improved the quality of kinetic images in 63.9% (2668 of 4176) of the comparisons. Interrater agreement was 75% and Fleiss k was 0.12 (P < .001). Also, 238 pairs of kinetic and DSA images were compared. Kinetic imaging was judged to have provided higher quality images than DSA in 69.0% (2462 of 3570) of the comparisons. The interrater agreement was 81% and Fleiss k was 0.17 (P < .001). Conclusion Kinetic imaging helps to view the same structures as digital subtraction angiography but offers better image quality. The improved signal-to-noise ratio suggests that this approach could reduce radiation exposure and improve the ability to view smaller vessels. © RSNA, 2018 Online supplemental material is available for this article.

Label-free Multiscale Transport Imaging of the Living Cell.

The living cell is characterized by a myriad of parallel intracellular transport processes. Simultaneously capturing their global features across multiple temporal and spatial scales is a nearly unsurmountable task. Here we present a method that enables the microscopic imaging of the entire spectrum of intracellular transport on a broad time scale without the need for prior labeling. We show that from the time-dependent fluctuation of pixel intensity, in either bright-field or phase-contrast microscopic images, a scaling factor can be derived that reflects the local Hurst coefficient (H), the value of which reveals the microscopic mechanisms of intracellular motion. The Hurst coefficient image of the interphase cell displays an unexpected, overwhelming superdiffusion (H > 0.5) in the cytoplasm and subdiffusion (H < 0.5) in the nucleus, and provides unprecedented sensitivity in detecting transport processes associated with the living state.

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.

Initial Experience Using Digital Variance Angiography in Context of Prostatic Artery Embolization in Comparison with Digital Subtraction Angiography.

RATIONALE AND OBJECTIVES: In previous clinical studies digital variance angiography (DVA) provided higher contrast-to-noise ratio (CNR) and better image quality in lower extremity angiography than digital subtraction angiography (DSA). Our aim was to investigate whether DVA has similar quality reserve in prostatic artery embolization (PAE). The secondary aim was to explore the potential advantages of the color-coded DVA (ccDVA) technology in PAE. MATERIAL AND METHODS: This retrospective study evaluated 108 angiographic acquisitions from 30 patients (mean ± SD age 68.0 ± 8.9, range 41-87) undergoing PAE between May and October 2020. DSA and DVA images were generated from the same unsubtracted acquisition, and their CNR was calculated. Visual evaluation of DVA and DSA image quality was performed by four experienced interventional radiologists in a randomized, blinded manner. The diagnostic value of DSA and ccDVA images was also evaluated using clinically relevant criteria (visibility of small [< 2.5 mm] and large arteries [> 2.5 mm], feeding arteries and tissue blush) in a paired comparison. Data were analysed by the Wilcoxon signed rank test or the binomial test, the interrater agreement was determined by the Kendall W or Fleiss Kappa analysis. RESULTS: DVA provided 4.11 times higher median CNR than DSA (IQR: 1.72). The visual score of DVA images (4.40 ± 0.05) was significantly higher than that of DSA (3.39 ± 0.07, p < 0.001). The Kendall W analysis showed moderate but significant agreement (WDVA = 0.38, WDSA = 0.53). The preference of ccDVA images was significantly higher in all criteria (63-89%) with an interrater agreement of 58-79%. The Fleiss Kappa range was 0.02-0.18, significant in all criteria except large vessels. CONCLUSION: Our data show that DVA provides higher CNR and better image quality in PAE. This quality reserve might be used for dose management (reduction of radiation dose and contrast agent volume), and ccDVA technology has also a high potential to assist PAE interventions in the future.

Radiation Exposure Reduction by Digital Variance Angiography in Lower Limb Angiography: A Randomized Controlled Trial.

BACKGROUND: digital variance angiography (DVA) provides higher image quality than digital subtraction angiography (DSA). This study investigates whether the quality reserve of DVA allows for radiation dose reduction during lower limb angiography (LLA), and compares the performance of two DVA algorithms. METHODS: this prospective block-randomized controlled study enrolled 114 peripheral arterial disease patients undergoing LLA into normal dose (ND, 1.2 µGy/frame, n = 57) or low-dose (LD, 0.36 µGy/frame, n = 57) groups. DSA images were generated in both groups, DVA1 and DVA2 images were generated in the LD group. Total and DSA-related radiation dose area product (DAP) were analyzed. Image quality was assessed on a 5-grade Likert scale by six readers. RESULTS: the total and DSA-related DAP were reduced by 38% and 61% in the LD group. The overall visual evaluation scores (median (IQR)) of LD-DSA (3.50 (1.17)) were significantly lower than the ND-DSA scores (3.83 (1.00), p < 0.001). There was no difference between ND-DSA and LD-DVA1 (3.83 (1.17)), but the LD-DVA2 scores were significantly higher (4.00 (0.83), p < 0.01). The difference between LD-DVA2 and LD-DVA1 was also significant (p < 0.001). CONCLUSIONS: DVA significantly reduced the total and DSA-related radiation dose in LLA, without affecting the image quality. LD-DVA2 images outperformed LD-DVA1, therefore DVA2 might be especially beneficial in lower limb interventions.

Possible use of Digital Variance Angiography in Liver Transarterial Chemoembolization: A Retrospective Observational Study.

PURPOSE: Digital variance angiography (DVA), a recently developed image processing technology, provided higher contrast-to-noise ratio (CNR) and better image quality (IQ) during lower limb interventions than digital subtraction angiography (DSA). Our aim was to investigate whether this quality improvement can be observed also during liver transarterial chemoembolization (TACE). MATERIALS AND METHODS: We retrospectively compared the CNR and IQ parameters of DSA and DVA images from 25 patients (65% male, mean ± SD age: 67.5 ± 11.2 years) underwent TACE intervention at our institute. CNR was calculated on 50 images. IQ of every image set was evaluated by 5 experts using 4-grade Likert scales. Both single image evaluation and paired image comparison were performed in a blinded and randomized manner. The diagnostic value was evaluated based on the possibility to identify lesions and feeding arteries. RESULTS: DVA provided significantly higher CNR (mean CNRDVA/CNRDSA was 1.33). DVA images received significantly higher individual Likert score (mean ± SEM 3.34 ± 0,08 vs. 2.89 ± 0.11, Wilcoxon signed-rank p < 0.001) and proved to be superior also in paired comparisons (median comparison score 1.60 [IQR:2.40], one sample Wilcoxon p < 0.001 compared to equal quality level). DSA could not detect lesion and feeding artery in 28 and 36% of cases, and allowed clear detection only in 22% and 16%, respectively. In contrast, DVA failed only in 8 and 18% and clearly revealed lesions and feeding arteries in 32 and 26%, respectively. CONCLUSION: In our study, DVA provided higher quality images and better diagnostic insight than DSA; therefore, DVA could represent a useful tool in liver TACE interventions. LEVEL OF EVIDENCE: III Non-consecutive study.

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.

Digital variance angiography allows about 70% decrease of DSA-related radiation exposure in lower limb X-ray angiography.

Our aim was to investigate whether the previously observed higher contrast-to-noise ratio (CNR) and better image quality of Digital Variance Angiography (DVA) - compared to Digital Subtraction Angiography (DSA) - can be used to reduce radiation exposure in lower limb X-ray angiography. This prospective study enrolled 30 peripheral artery disease patients (mean ± SD age 70 ± 8 years) undergoing diagnostic angiography. In all patients, both normal (1.2 µGy/frame; 100%) and low-dose (0.36 µGy/frame; 30%) protocols were used for the acquisition of images in three anatomical regions (abdominal, femoral, crural). The CNR of DSA and DVA images were calculated, and the visual quality was evaluated by seven specialists using a 5-grade Likert scale. For investigating non-inferiority, the difference of low-dose DVA and normal dose DSA scores (DVA30-DSA100) was analyzed. DVA produced two- to three-fold CNR and significantly higher visual score than DSA. DVA30 proved to be superior to DSA100 in the crural region (difference 0.25 ± 0.07, p < 0.001), and there was no significant difference in the femoral (- 0.08 ± 0.06, p = 0.435) and abdominal (- 0.10 ± 0.09, p = 0.350) regions. Our data show that DVA allows about 70% reduction of DSA-related radiation exposure in lower limb X-ray angiography, providing a potential new radiation protection tool for the patients and the medical staff.

Initial Operating Room Experience with Digital Variance Angiography in Carbon Dioxide-Assisted Lower Limb Interventions: A Pilot Study.

PURPOSE: In retrospective clinical studies digital variance angiography (DVA) provided higher contrast-to-noise ratio and better image quality than digital subtraction angiography (DSA). Our aim was to verify the clinical usefulness and benefits of DVA in carbon dioxide (CO2)-assisted lower limb interventions. MATERIALS AND METHODS: A workstation running the DVA software was integrated into a Siemens Artis Zee with Pure angiography system, and this new image processing technology was used in four patients (3 male, 1 female, age: 76.2 ± 4.2 years) with peripheral artery disease (PAD, Rutherford 2-3) and impaired renal function (average eGFR 25.5 ± 11.2 ml/min/1.73 m2). The DSA and DVA images of 46 CO2-assisted runs were visually evaluated by five experts in single-image evaluation using a 5-grade Likert scale and in paired comparisons. RESULTS: DVA images received significantly higher score (3.84 ± 0.10) than DSA images (3.31 ± 0.10, p < 0.001). Raters preferred DVA images in terms of diagnostic value and usefulness for therapeutic decisions in 85.2% and 83.9% of all comparisons, respectively. These benefits were achieved at lower frame rates (1-3 FPS) than usually recommended for CO2 angiography (4-6 FPS). No adverse events were recorded during or after the procedures. CONCLUSIONS: Our initial experience shows that DVA might facilitate the correct diagnostic and therapeutic decisions, and potentially help to reduce radiation exposure in lower limb CO2 angiography. Although the dose management capabilities of DVA have to be validated in further clinical studies, this technology might be a useful new tool in the operating room and contributes to the safety and efficacy of CO2-enhanced endovascular interventions. LEVEL OF EVIDENCE: Level IV.

Initial evidence of a 50% reduction of contrast media using digital variance angiography in endovascular carotid interventions.

PURPOSE: In previous clinical studies Digital Variance Angiography (DVA) provided higher signal-to-noise ratio (SNR) and better image quality than Digital Subtraction Angiography (DSA). Our aim was to investigate whether this quality reserve of DVA provides an opportunity for the reduction of iodinated contrast media (ICM) in carotid X-ray angiography (CXA). METHOD: Our prospective study enrolled 26 patients (67.0 ±â€¯8.1 years) undergoing carotid percutaneous transluminal angioplasty. The SNR of DSA and DVA image pairs obtained by a standard (100 %, 6 mL ICM) or a low-dose (50 %, 3 mL ICM) protocol were compared. Visual evaluation of all images was performed by five specialists using a 5-grade rating scale. The quality of DSA100 and DVA50 videos was also compared. RESULTS: DVA provided more than two-fold SNR, the median SNRDVA/SNRDSA ratio was 2.06 (100 %) and 2.25 (50 %). In the visual evaluation, the DVA100 score (3.73 ±â€¯0.06) was significantly higher than the DSA100 score (3.52 ±â€¯0.07, Wilcoxon p < 0.001), and the DVA50 score (3.64 ±â€¯0.13) was also significantly higher than the DSA50 score (3.01 ±â€¯0.17, Wilcoxon p < 0.001). While the low-dose protocol significantly decreased the DSA score (Mann-Whitney p < 0.01, DSA100 vs DSA50), it had no effect on the DVA score (DVA100 vs DVA50). There was no statistical difference between the DSA100 and DVA50 scores. Evaluators preferred the diagnostic value of DVA50 to DSA100 videos in 61% of comparisons, the interrater agreement was 69 % (Fleiss' kappa 0.35, p < 0.001). CONCLUSIONS: Our data show that DVA allows a substantial (50 %) ICM reduction in CXA without affecting the quality and diagnostic value of angiograms.

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.

The structure of horseradish peroxidase C characterized as a molten globule state after Ca(2+) 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.

Digital Variance Angiography as a Paradigm Shift in Carbon Dioxide Angiography.

OBJECTIVES: Our aim was to investigate the feasibility of digital variance angiography (DVA) in lower extremity CO2 angiography and to compare the quantitative and qualitative performance of the new image processing technique with that of the current reference standard digital subtraction angiography (DSA). MATERIALS AND METHODS: This prospective study enrolled 24 patients (mean age ± SD, 65.5 ± 9.2 years; 14 males, 65.1 ± 7.5 years; 10 females, 66.1 ± 11.6 years) undergoing lower-limb CO2 angiography between December 2017 and April 2018 at 2 clinical centers: The Heart and Vascular Center (HVC) of Semmelweis University, Budapest (7 patients), and the Bács-Kiskun County Hospital (BKCH) in Kecskemét (17 patients). The interventional protocol was similar at both sites, but the image acquisition instruments and protocols were different, which allowed us to investigate DVA in different settings. For comparison, the signal-to-noise ratio (SNR) of DSA and DVA images were calculated. The visual quality of DSA and DVA images were compared by independent clinical specialists using an online questionnaire. Interrater agreement was characterized by percent agreement and Fleiss kappa. The specialists also evaluated in a random and blinded manner the individual DSA and DVA images on a 5-grade scale ranging from poor (1) to outstanding (5) image quality, and the mean ± standard error of mean (SEM) was calculated. RESULTS: A total of 4912 regions of interest were carefully selected in 110 image pairs to determine the SNRs. The ratio of SNRDVA/SNRDSA was calculated. At HVC, it ranged between 2.58 and 4.16 in the anatomical regions (abdominal, iliac, femoral, popliteal, crural, talar), and the overall median value was 3.53, whereas at BKCH the range was 2.71 to 4.92 and the overall median value was 4.52. During the visual evaluation, 120 DSA and DVA image pairs were compared. At HVC in 78%, although at BKCH in 90% of comparisons, it was judged that DVA provided higher quality images. The interrater agreement was 88% (P < 0.001) and 90% (P < 0.01), respectively. DVA images received consistently higher individual rating than DSA images, regardless of the research site and anatomical region. At HVC, the overall DSA and DVA scores (mean ± SEM) were 2.75 ± 0.12 and 3.23 ± 0.16, respectively (P < 0.05), whereas at BKCH these values were 2.49 ± 0.10 and 3.03 ± 0.09, respectively (P < 0.001). CONCLUSIONS: These data show that lower-limb CO2 angiography DVA, regardless of the image acquisition instruments and protocols, produces higher SNR and significantly better image quality than DSA; therefore this new image processing technique might help the widespread use of CO2 as a safer contrast agent in clinical practice.

Determination of long-lived Nb isotopes in nuclear power plant wastes.

(94)Nb and (93m)Nb are long-lived radionuclides, produced by thermal and fast neutrons from (93)Nb that is a major component of the Zr alloys used in nuclear reactors. A radiochemical method for the determination of these nuclides has been developed. The separation is based on the insolubility of Nb oxides and the retention of the fluoric complexes on anion exchange resin. The Nb sources are detected by gamma- and X-ray spectrometries. Activity concentrations determined in radioactive waste samples of a nuclear power plant are presented.

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.

Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase.

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. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60-100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.

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.

Tuning the heterogeneous early folding dynamics of phosphoglycerate kinase.

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.