Modification of the thermal unfolding pathways of myoglobin upon drug interaction in different aqueous media

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.     

Different thermal unfolding pathways of catalase in the presence of cationic surfactants

In this paper we have corroborated the usefulness of spectroscopic techniques, such as UV-visible, in the study and thermodynamic characterization of the thermal unfolding of catalase as a function of the concentration and alkyl chain length of n-alkyltrimethylammonium bromides (CnTAB, n = 8, 10, and 12). For this reason, a thermodynamic model was used which included experimental data corresponding to the pre- and posttransition into the observable transition. It has been found that n-alkyltrimethylammonium bromides play two opposite roles in the folding and stability of catalase. They act as a structure stabilizer at a low molar concentration and as a destabilizer at a higher concentration. The maximum of the unfolding temperature has been found to decrease with the alkyl chain. The reason for this difference has been suggested to be the side chains involved. In the presence of C8TAB and C10TAB, Gibbs energies of unfolding (DeltaG(T)) decrease with concentration, whereas for C12TAB an increase has been observed. These findings can be explained by the fact that when differences in the hydrophobic nature of the surfactants exist, different pathways of unfolding may occur. Also, the presence of surfactants has been observed to affect the cold denaturation of catalase. Thermodynamic results suggest that the thermal denaturation of catalase in the presence of n-alkyltrimethylammonium bromides is a perfect transition between two states.     

Application of the localized representation for studying interaction energies

It has already been shown that the use of the localized many-body perturbation theory (LMBPT ) makes its possible to calculate the interaction energy at the correlated level in a straightfoward way. In this article, we show that the correlated part of the interaction energy, furthermore, can simply be decomposed into dispersion and charge-transfer contributions using the LMBPT scheme. The CH₂O + NH₃ model system was chosen for the above study. © 1996 John Wiley & Sons, Inc.     

Stretching single molecules along unbinding and unfolding pathways with the scanning force microscope

Individual molecules can be stretched with a scanning force microscope and the forces required to rupture bonds and to mechanically drive their structures towards new conformations and states can be measured. The tailoring of the experiments, the possibility of carrying them out in quasi-equilibrium conditions, the relationships between single molecule force measurements, and macroscopic kinetics or thermodynamic data are discussed. Mechanochemical experiments are expanding chemistry into new realms between biology and material science.     

Simultaneous characterization of the reductive unfolding pathways of RNase B isoforms by top-down mass spectrometry

A novel method for characterization of the simultaneous reductive unfolding pathways of five isoforms of bovine pancreatic ribonuclease B (RNase B) is demonstrated. The results indicate that each isoform unfolds reductively through two three-disulfide-containing structured intermediates before proceeding to the fully reduced form, as in the reductive unfolding pathways of the A variant lacking the carbohydrate chain. The rates of reduction of bovine pancreatic ribonuclease A (RNase A) and RNase B and the formation and consumption of their reductive intermediates are identical, indicating that the unfolding events necessary to expose disulfide bonds for reduction are not affected by the oligosaccharide. The method utilizes top-down mass spectrometry and a naturally occurring tag on the protein, viz. the carbohydrate moiety, to obtain unfolding information of an ensemble of protein isoforms and is a generally applicable methodological advance for conducting folding studies on mixtures of different proteins.     

Evidence of an intermediate and parallel pathways in protein unfolding from single-molecule fluorescence

Determining how proteins fold into their native structures is a subject of great importance, since ultimately it will allow protein structure and function to be predicted from primary sequence data. In addition, there is now a clear link between protein unfolding and misfolding events and many disease states. However, since proteins fold over rugged, multidimensional energy landscapes, this is a challenging experimental and theoretical problem. Single-molecule fluorescence methods developed over the past decade have the potential to follow the unfolding/folding of individual molecules. Mapping out the landscape without ensemble averaging will enable the identification of intermediate states which may not be significantly populated, in addition to the presence of multiple pathways. To date, there have been only a limited number of single-molecule folding/unfolding studies under nonequilibrium conditions and no intermediates have been observed. Here, for the first time, we present a single-molecule study of the unfolding of a large autofluorescent protein, Citrine, a variant of green fluorescent protein. Single-molecule fluorescence techniques are used to directly detect an intermediate on the unfolding/folding pathway and the existence of parallel unfolding pathways. This work, and the novel methods used, shows that single-molecule fluorescence can now provide new, hitherto experimentally inaccessible, insights into the folding/unfolding of proteins.     

Symmetry of localized orbitals in molecules with spin-orbit interaction

We construct representations of crystallographic point groups induced by double-valued irreducible representations of their local subgroups. Using the tetrahedral molecule Bi₄ as an example, we illustrate application of the results obtained to the symmetry analysis of localized molecular orbitals obtained from relativistic calculations.Translated from Teoreticheskaya i éksperimental'naya Khimiya, Vol. 24, No. 1, pp. 91–95, January–February, 1988.     

A facile synthesis of the lowest homologues of meso-tetraalkylporphyrin

Utilization of a less stoichiometric amount of aldehyde in portions and addition of water to the reaction mixture depressed the tar formation, affording much easier isolation of meso-tetraalkylporphyrins. The title porphyrins bearing methyl, ethyl, or n-propyl substituents were synthesized in isolated yields of 10,10, and 8%, respectively.     

Magnetic tweezers measurement of the bond lifetime-force behavior of the IgG-protein A specific molecular interaction

The bond lifetime-force behavior of the immunoglobulin G (IgG)-protein A interaction has been studied with magnetic tweezers to characterize the physical properties of the bond under nonequilibrium conditions. Super-paramagnetic microparticles were developed that have a high and uniform magnetization to simultaneously apply a piconewton-scale tensile force to many thousands of IgG-protein A bonds. A strong and a weak slip bond were detected with an effective bond length that is characteristic of short-range, stiff intermolecular interactions. These bonds are attributed to the interaction of protein A with the constant region (Fc) and heavy chain variable domain (VH) of IgG, respectively. The IgG-VH interaction appears to be one of the weakest specific molecular interactions that has been identified with a single molecule force measurement technique. This study demonstrates that magnetic tweezers can be used to rapidly characterize very weak biomolecular interactions as well as strong biomolecular interactions with a high degree of accuracy.     

A note on observability of fluctuation-induced structural interaction in a nematic mesophase

We discuss the observability of pseudo-Casimir interaction in nematic liquid crystals, and show that in physically relevant regimes the interaction is characterized by non-universal force profiles that depend strongly on the anchoring. Depending on the ratio of the anchoring strengths, the force may be either purely attractive at all separations or it may become repulsive at separations comparable to the geometric mean of the extrapolation lengths. Within the currently accessible experimental window, it appears that the 1/h ³ force could only be seen in very weakly anchored symmetric systems at separations no larger than a few 10 nm. We also find that while the conventional force measuring systems such as the atomic force microscope and surface force apparatus can provide some information on the fluctuationinduced force, alternative techniques, e.g. spinodal dewetting, could be used to obtain a more comprehensive insight extending over a wider range of separations.     

A note on observability of fluctuation-induced structural interaction in a nematic mesophase

We discuss the observability of pseudo-Casimir interaction in nematic liquid crystals, and show that in physically relevant regimes the interaction is characterized by non-universal force profiles that depend strongly on the anchoring. Depending on the ratio of the anchoring strengths, the force may be either purely attractive at all separations or it may become repulsive at separations comparable to the geometric mean of the extrapolation lengths. Within the currently accessible experimental window, it appears that the 1/h ³ force could only be seen in very weakly anchored symmetric systems at separations no larger than a few 10 nm. We also find that while the conventional force measuring systems such as the atomic force microscope and surface force apparatus can provide some information on the fluctuationinduced force, alternative techniques, e.g. spinodal dewetting, could be used to obtain a more comprehensive insight extending over a wider range of separations.     

Contrasting the individual reactive pathways in protein unfolding and disulfide bond reduction observed within a single protein

Identifying the dynamics of individual molecules along their reactive pathways remains a major goal of modern chemistry. For simple chemical reactions, the transition state position is thought to be highly localized. Conversely, in the case of more complex reactions involving proteins, the potential energy surfaces become rougher, resulting in heterogeneous reaction pathways with multiple transition state structures. Force-clamp spectroscopy experimentally probes the individual reaction pathways sampled by a single protein under the effect of a constant stretching force. Herein, we examine the distribution of conformations that populate the transition state of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. By applying the recently developed static disorder theory, we quantify the variance of the barrier heights, σ(2), governing each distinct reaction. We demonstrate that the unfolding of the I27 protein follows a nonexponential kinetics, consistent with a high value of σ(2) ～ 18 (pN nm)(2). Interestingly, shortening of the protein upon introduction of a rigid disulfide bond significantly modulates the disorder degree, spanning from σ(2) ～ 8 to ～21 (pN nm)(2). These results are in sharp contrast with the exponential distribution of times measured for an S(N)2 chemical reaction, implying the absence of static disorder σ(2) ～ 0 (pN nm)(2). Our results demonstrate the high sensitivity of the force-clamp technique to capture the signatures of disorder in the individual pathways that define two distinct force-induced reactions, occurring within the core of a single protein.     

Integrated prediction of protein folding and unfolding rates from only size and structural class

Protein stability, folding and unfolding rates are all determined by the multidimensional folding free energy surface, which in turn is dictated by factors such as size, structure, and amino-acid sequence. Work over the last 15 years has highlighted the role of size and 3D structure in determining folding rates, resulting in many procedures for their prediction. In contrast, unfolding rates are thought to depend on sequence specifics and be much more difficult to predict. Here we introduce a minimalist physics-based model that computes one-dimensional folding free energy surfaces using the number of aminoacids (N) and the structural class (α-helical, all-β, or α-β) as only protein-specific input. In this model N sets the overall cost in conformational entropy and the net stabilization energy, whereas the structural class defines the partitioning of the stabilization energy between local and non-local interactions. To test its predictive power, we calibrated the model empirically and implemented it into an algorithm for the PREdiction of Folding and Unfolding Rates (PREFUR). We found that PREFUR predicts the absolute folding and unfolding rates of an experimental database of 52 proteins with accuracies of ±0.7 and ±1.4 orders of magnitude, respectively (relative to experimental spans of 6 and 8 orders of magnitude). Such prediction uncertainty for proteins vastly varying in size and structure is only two-fold larger than the differences in folding (±0.34) and unfolding rates (±0.7) caused by single-point mutations. Moreover, PREFUR predicts protein stability with an accuracy of ±6.3 kJ mol(-1), relative to the 5 kJ mol(-1) average perturbation induced by single-point mutations. The remarkable performance of our simplistic model demonstrates that size and structural class are the major determinants of the folding landscapes of natural proteins, whereas sequence variability only provides the final 10-20% tuning. PREFUR is thus a powerful bioinformatic tool for the prediction of folding properties and analysis of experimental data.     

Identifying cancer specific signaling pathways based on the dysregulation between genes

A large collection of studies has shown that the occurrence of cancer is related to the functional dysfunction of the pathways. Identification of cancer-related pathways could help researchers understand the mechanisms of complex diseases well. Whereas, most current signaling pathway analysis methods take no account of the gene interaction variations within pathways. Furthermore, considering that some pathways have connection with two or more cancer types, while some are likely to be cancer-type specific pathways. Identifying cancer-type specific pathways contributes to interpreting the different mechanisms of different cancer types. In this study, we first proposed a pathway analysis method named Pathway Analysis of Intergenic Regulation (PAIGR) to identify pathways with dysregulation between genes and compared the performance of this method with four existing methods on four colorectal cancer (CRC) datasets. The results showed that PAIGR could find cancer-related pathways more accurately. Moreover, in order to explore the relationship between the identified pathways and the cancer type, we constructed a pathway interaction network, in which nodes and edges represented pathways and interactions between pathways respectively. Highly connected pathways were considered to play a central role in an extensive range of biological processes, while sparsely connected pathways are considered to have certain specificity. Our results showed that pathways identified by PAIGR had a low nodal degree (i.e., a few numbers of interactions), which suggested that most of these pathways were cancer-type specific.     

Reinvestigation of the reactions of camphorketene: structural evidence for pseudopericyclic pathways

The stereochemistry of the dimers (3 and 4) of camphorketene (2) have been determined. The crystal structures of 3, 20 and of related compounds show ground-state distortions that are interpreted as prefiguring planar, pseudopericyclic transition states for retro-cycloadditions to form alpha-oxoketenes. The B3LYP/6-31G* optimized geometry for the transition structure (10) for the dimerization of s-Z-formylketene (8) is consistent with this mechanism. Trapping of 2 with alcohols shows selectivity comparable to other alpha-oxoketenes. The lack of reaction of 2 with benzaldehyde and the lack of enol tautomers in camphoric acid derivatives is attributed to angle strain in the bicyclic camphor moiety.     

Influence of the sample-solvent on protein retention,mass transfer and unfolding kinetics in hydrophobic interaction chromatography

Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption–desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed.     

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, α-chymotrypsinogen A, β-lactoglobulin B, and holo α-lactalbumin, are studied on Sepharose™ HIC resins possessing assorted ligand chemistries and densities. α-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed β-lactoglobulin B and holo α-lactalbumin range from −4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2–0.9 min⁻¹. While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.     

A theoretical study on the specific interaction of hexafluorobenzene with benzene and p -xylene

A cndo/2 study has been carried out for C₆F₆ + C₆H₆aad C₆F₆ + C₆H₁₀ composites and individual molecules. The favoured configurations of the adducts have been decided on the basis of energy calculations of various geometries. For the C₆F₆+C₆H₆ adduct the lowest energy corresponds to the configuration in which the molecular planes are parallel to each other with a twist angle of 30°. For the C₆F₆+ C₈H₁₀ adduct lowest energy corresponds to a geometry in which the two molecular planes are inclined by a small angle with the angle of twist between the molecular planes being 30°. It is shown that the complexes are not of the charge transfer type.