Nonadditivity in the alpha-helix to coil transition

The Lifson-Roig Model (LRM) and all its variants describe the α-helix to coil transition in terms of additive component-free energies within a free energy decomposition scheme, and these contributions are interpreted through sequence-context dependent nucleation and propagation parameters. Although this phenomenological approach is able to adequately fit experimental data on helix content and heat capacity, the number of required parameters increases dramatically with additional sequence variation. Moreover, due to nonadditive competing microscopic effects that are difficult to disentangle within a LRM, large uncertainties within the parameters emerge. We offer an alternative view that removes the need for sequence-context parameterization by focusing on individual microsopic interactions within a free energy decomposition and explicitly account for nonadditivity in conformational entropy through network rigidity using a Distance Constraint Model (DCM). We apply a LRM and a DCM to previously published experimental heat capacity and helix content data for a series of heterogeneous polypeptides. Both models describe the experimental data well, and the parameters from both models are consistent with prior work. However, the number of DCM parameters is independent of sequence-variability, the parameter values exhibit better transferability, and the helix nucleation is predicted by the DCM explicitly through the nonadditive nature of conformational entropy. The importance of these results is that the DCM offers a system-independent approach for modeling stability within polypeptides and proteins, where the demonstrated accuracy for the α-helix to coil transition over a series of heterogeneous polypeptides described here is one case in point.     

Elucidating protein thermodynamics from the three-dimensional structure of the native state using network rigidity

Given the three-dimensional structure of a protein, its thermodynamic properties are calculated using a recently introduced distance constraint model (DCM) within a mean-field treatment. The DCM is constructed from a free energy decomposition that partitions microscopic interactions into a variety of constraint types, i.e., covalent bonds, salt-bridges, hydrogen-bonds, and torsional-forces, each associated with an enthalpy and entropy contribution. A Gibbs ensemble of accessible microstates is defined by a set of topologically distinct mechanical frameworks generated by perturbing away from the native constraint topology. The total enthalpy of a given framework is calculated as a linear sum of enthalpy components over all constraints present. Total entropy is generally a nonadditive property of free energy decompositions. Here, we calculate total entropy as a linear sum of entropy components over a set of independent constraints determined by a graph algorithm that builds up a mechanical framework one constraint at a time, placing constraints with lower entropy before those with greater entropy. This procedure provides a natural mechanism for enthalpy-entropy compensation. A minimal DCM with five phenomenological parameters is found to capture the essential physics relating thermodynamic response to network rigidity. Moreover, two parameters are fixed by simultaneously fitting to heat capacity curves for histidine binding protein and ubiquitin at five different pH conditions. The three free parameter DCM provides a quantitative characterization of conformational flexibility consistent with thermodynamic stability. It is found that native hydrogen bond topology provides a key signature in governing molecular cooperativity and the folding-unfolding transition.     

Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.     

Unifying mechanical and thermodynamic descriptions across the thioredoxin protein family

We compare various predicted mechanical and thermodynamic properties of nine oxidized thioredoxins (TRX) using a Distance Constraint Model (DCM). The DCM is based on a nonadditive free energy decomposition scheme, where entropic contributions are determined from rigidity and flexibility of structure based on distance constraints. We perform averages over an ensemble of constraint topologies to calculate several thermodynamic and mechanical response functions that together yield quantitative stability/flexibility relationships (QSFR). Applied to the TRX protein family, QSFR metrics display a rich variety of similarities and differences. In particular, backbone flexibility is well conserved across the family, whereas cooperativity correlation describing mechanical and thermodynamic couplings between the residue pairs exhibit distinctive features that readily standout. The diversity in predicted QSFR metrics that describe cooperativity correlation between pairs of residues is largely explained by a global flexibility order parameter describing the amount of intrinsic flexibility within the protein. A free energy landscape is calculated as a function of the flexibility order parameter, and key values are determined where the native‐state, transition‐state, and unfolded‐state are located. Another key value identifies a mechanical transition where the global nature of the protein changes from flexible to rigid. The key values of the flexibility order parameter help characterize how mechanical and thermodynamic response is linked. Variation in QSFR metrics and key characteristics of global flexibility are related to the native state X‐ray crystal structure primarily through the hydrogen bond network. Furthermore, comparison of three TRX redox pairs reveals differences in thermodynamic response (i.e., relative melting point) and mechanical properties (i.e., backbone flexibility and cooperativity correlation) that are consistent with experimental data on thermal stabilities and NMR dynamical profiles. The results taken together demonstrate that small‐scale structural variations are amplified into discernible global differences by propagating mechanical couplings through the H‐bond network. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

1,1,1,3,3,3-hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine alpha-lactalbumin: calorimetric and spectroscopic studies

The thermal denaturation of alpha-lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van't Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of alpha-lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that alpha-lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that alpha-lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of alpha-lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of alpha-lactalbumin.     

Revealing the nature of the native state ensemble through cold denaturation

Recent advances in NMR methodology have enabled the structural analysis of proteins at temperatures far below the freezing point of water, thus opening a window to the cold denaturation process. Although the phenomenon of cold denaturation has been known since the mid-1970s, the freezing point of water has prevented detailed and structurally resolved studies without application of additional significant perturbations of the protein ensemble. As a result, the cold-denatured state and the process of cold denaturation have gone largely unstudied. Here, the structural and thermodynamic basis of cold denaturation is explored with emphasis placed on the insights that are uniquely ascertained from low-temperature studies. It is shown that the noncooperative cold-induced unfolding of protein results in the population of partially folded states that cannot be accessed by other techniques. The structurally resolved view of the cold denaturation process therefore can provide direct access to the cooperative substructures within the protein molecule and provide an unprecedented structurally resolved picture of the states that comprise the native state ensemble.     

Powerful solvent systems useful for synthesis of sparingly-soluble peptides in solution.

Our maximum protection strategy for the synthesis of human parathyroid hormone(1-84) indicates that fully protected peptide segments in the form of Boc-peptide phenacyl (Pac) ester are relatively soluble in ordinary organic solvents such as DMF, NMP or DMSO, which are suitable for coupling segments. However, about 1% of such segments synthesized were found to be insoluble even in the most polar solvent, DMSO. Thus, a more powerful solvent which can be used for their peptide synthesis was pursued. Among the solvent systems tested, a mixture of trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) and trichloromethane (TCM) or dichloromethane (DCM) was found to be most powerful for dissolving such sparingly-soluble protected peptides. These solvent systems were confirmed to be useful for the removal reaction of the carboxy-terminal Pac esters from the sparingly-soluble segments. They were then tested for the coupling reactions of fully protected Boc-peptides with other sparingly-soluble peptide esters. The TFE/TCM or TFE/DCM system was extremely useful for coupling segments without danger of racemization and of trifluoroester formation, if WSCI was used as the coupling reagent in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt).     

Distinctive influence of two hexafluoro solvents on the structural stabilization of Bombyx mori silk fibroin protein and its derived peptides: 13C NMR and CD studies

Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent.     

Manipulation of individual viruses: friction and mechanical properties.

We present our results on the manipulation of individual viruses using an advanced interface for atomic force microscopes (AFMs). We show that the viruses can be dissected, rotated, and translated with great facility. We interpret the behavior of tobacco mosaic virus with a mechanical model that makes explicit the competition between sample-substrate lateral friction and the flexural rigidity of the manipulated object. The manipulation behavior of tobacco mosaic virus on graphite is shown to be consistent with values of lateral friction observed on similar interfaces and the flexural rigidity expected for macromolecular assemblies. The ability to manipulate individual samples broadens the scope of possible studies by providing a means for positioning samples at specific binding sites or predefined measuring devices. The mechanical model provides a framework for interpreting quantitative measurements of virus binding and mechanical properties and for understanding the constraints on the successful, nondestructive AFM manipulation of delicate samples.     

Calculating ensemble averaged descriptions of protein rigidity without sampling

Previous works have demonstrated that protein rigidity is related to thermodynamic stability, especially under conditions that favor formation of native structure. Mechanical network rigidity properties of a single conformation are efficiently calculated using the integer body-bar Pebble Game (PG) algorithm. However, thermodynamic properties require averaging over many samples from the ensemble of accessible conformations to accurately account for fluctuations in network topology. We have developed a mean field Virtual Pebble Game (VPG) that represents the ensemble of networks by a single effective network. That is, all possible number of distance constraints (or bars) that can form between a pair of rigid bodies is replaced by the average number. The resulting effective network is viewed as having weighted edges, where the weight of an edge quantifies its capacity to absorb degrees of freedom. The VPG is interpreted as a flow problem on this effective network, which eliminates the need to sample. Across a nonredundant dataset of 272 protein structures, we apply the VPG to proteins for the first time. Our results show numerically and visually that the rigidity characterizations of the VPG accurately reflect the ensemble averaged [Formula: see text] properties. This result positions the VPG as an efficient alternative to understand the mechanical role that chemical interactions play in maintaining protein stability.     

Hairpin folding dynamics: the cold-denatured state is predisposed for rapid refolding

Cold denaturation is a general phenomenon in globular proteins, and the associated cold-denatured states of proteins have important fundamental and practical significance. Here, we have characterized the cold-denatured state of a beta-hairpin forming peptide, MrH3a, in 8% hexafluoro-2-propanol (HFIP) and the dynamics of its refolding following a laser-induced T-jump. Beta-hairpins constitute an important class of protein structural elements, yet their folding mechanisms are not fully understood. Characterization of MrH3a using NMR, CD, and IR spectroscopies reveals residual structure in the cold-denatured state, in contrast with the highly disordered heat-denatured state. The residual structure in the cold-denatured state comprises relatively compact and solvent protected conformations. Furthermore, we find a substantial acceleration in the rate of folding from the cold-denatured state compared to that of the heat-denatured state. In addition, the cold-denatured state is not populated in 20% HFIP; folding occurs only from the fully unfolded state and is significantly slower. We interpret the acceleration of the folding rate of MrH3a in 8% HFIP as a direct consequence of the collapsed conformations of the cold-denatured state. Finally, there may be some reduction of the loop search cost when starting from the cold-denatured state, since this state may have some of the stabilizing cross-strand interactions already formed.     

Protein folding and wring resonances

The polypeptide chain of a protein is shown to obey topological constraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding of proteins) are natural consequences of the suggested wring mode model. Native (folded) proteins are found to possess an intrinsic standing wring mode.     

A comparative thermodynamic study of heat and cold denaturation of beta-lactoglobulin]

The changes in structure and thermodynamic parameters of beta-lactoglobulin upon heat and cold denaturation have been studied using both scanning microcalorimetry and circular dichroism spectroscopy methods. It has been shown that in contrast to the heat denaturation process, the cold denaturation of beta-lactoglobulin is accompanied by an opposite heat effect. In all cases, the calorimetrically measured enthalpy of beta-lactoglobulin cold denaturation is higher than it was expected from the two-state model of denaturation transition. It has been concluded that beta-lactoglobulin cold denaturation cannot be represented by a transition between two microscopic states--native and denatured. The latter, is due to the additional process that occurs together with the disruption of the beta-lactoglobulin tertiary structure and is accompanied by increasing heat capacity. Taking into account the heat capacity contribution of this process upon calculation of the enthalpy makes it closer to the enthalpy value calculated for the two-state model of denaturation transition.     

Volatile Anesthetic Sevoflurane Precursor 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) Exerts an Anti-Prion Activity in Prion-Infected Culture Cells

Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrP^C) to the abnormal β-sheet rich form (PrP^Sc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrP^Sc, reduced PrP^Sc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease.

     

Linking membrane physical properties and low temperature tolerance in arthropods

Maintenance of membrane fluidity is of crucial importance in ectotherms experiencing thermal changes. This maintenance has in ectotherms most often been indicated using indirect measures of biochemical changes of phospholipid membranes, which is then assumed to modulate the physico-chemical properties of the membrane. Here, we measure bending rigidity characterizing the membrane flexibility of re-constituted membrane vesicles to provide a more direct link between membrane physical characteristics and low temperature tolerance. Bending rigidity of lipid bilayers was measured in vitro using Giant Unilamellar Vesicles formed from phospholipid extracts of the springtail, Folsomia candida. The bending rigidity of these membranes decreased when exposed to 0.4 vol% ethanol (0.23 mM/L). Springtails exposed to ethanol for 24 h significantly increased their cold shock tolerance. Thus, by chemically inducing decreased membrane rigidity, we have shown a direct link between the physico-chemical properties of the membranes and the capacity to tolerate low temperature in a chill-susceptible arthropod.     

Analyzing the flexibility of RNA structures by constraint counting

RNA requires conformational dynamics to undergo its diverse functional roles. Here, a new topological network representation of RNA structures is presented that allows analyzing RNA flexibility/rigidity based on constraint counting. The method extends the FIRST approach, which identifies flexible and rigid regions in atomic detail in a single, static, three-dimensional molecular framework. Initially, the network rigidity of a canonical A-form RNA is analyzed by counting on constraints of network elements of increasing size. These considerations demonstrate that it is the inclusion of hydrophobic contacts into the RNA topological network that is crucial for an accurate flexibility prediction. The counting also explains why a protein-based parameterization results in overly rigid RNA structures. The new network representation is then validated on a tRNA^ASP structure and all NMR-derived ensembles of RNA structures currently available in the Protein Data Bank (with chain length ≥40). The flexibility predictions demonstrate good agreement with experimental mobility data, and the results are superior compared to predictions based on two previously used network representations. Encouragingly, this holds for flexibility predictions as well as mobility predictions obtained by constrained geometric simulations on these networks. Potential applications of the approach to analyzing the flexibility of DNA and RNA/protein complexes are discussed.     

Simple assay of plasma sevoflurane and its metabolite hexafluoroisopropanol by headspace GC–MS

The anesthetic sevoflurane can now be delivered over periods of up to 48 h using a newly developed medical system, the AnaConDa (anesthetic conserving device). Lack of pharmacokinetic data on sevoflurane and its main metabolite (hexafluoroisopropanol, HFIP) in this indication prompted us to develop a headspace GC–MS method to quantify the two substances. The only previously published method for assaying the two substances could not be adapted to our study since it uses expensive and rarely employed system components together with toxic carbon disulfide as a dilution solvent. The method developed is straightforward and uses the relatively non-toxic solvent undecane as dilution solvent and chloroform as internal standard. The method is linear for a concentration range of 1–150 μg/ml, and presents high accuracy and precision. LOD and LOQ are 0.2 and 1 μg/ml, with a short analysis time (7.6 min for a single analysis). The method was applied to determine the plasma levels of sevoflurane and HFIP in six patients under 48-h anesthetic sedation delivered via the AnaConDa system. Average sevoflurane and HFIP concentrations plateaued at 75 and 4 μg/ml, respectively. Sevoflurane quickly tailed off after inhalation was stopped, and HFIP levels remained low.     

Observation of Solvent Penetration during Cold Denaturation of E.?coli Phosphofructokinase-2

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N₂ 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.     

Biophysical models of protein denaturation. II. Effects of denaturants and of pH.

In order to broaden the scope and increase the utility of differential scanning calorimetry, a theoretical model of calorimetric thermograms is presently proposed which facilitates their biophysical interpretation and accounts explicitly for their modifications induced by denaturing agents and/or pH. The model rests mainly on statistical-physical considerations, the denaturation-linked increase of the number of binding sites for denaturants (including H+) serving as the conceptual basis for thermogram modelling. Denaturants were envisioned as contributing indirectly to thermal denaturation by forming complexes preferentially with unfolded protein molecules, shifting thus the equilibrium towards the denatured phase. After postulating the probability of complex formation, mean numbers of the relevant molecular species were computed by ensemble averaging. Finally, an eight-parameter expression has been derived defining protein heat capacity as a function of both temperature and denaturant concentration (or pH), each of the eight parameters having a distinct biophysical meaning. The model has been tested by applying it to the prediction of the pH-dependence of thermograms. Four proteins have been considered (lysozyme, myoglobin, apomyoglobin, and ribonuclease A), each represented by a series of three to four published thermograms recorded under different pH conditions. Model equations, fitted simultaneously to all thermograms in a pH series, reproduced correctly experimental tracings. Parameter values obtained as best-fit requirements (particularly those representing the number of binding sites unmasked by denaturation and the free energy of ion binding) were in close agreement with empirical, mainly potentiometric, data from literature. The empirically established pH-independence of the total enthalpy of denaturation, the phenomenon of cold denaturation, the pH-dependence of the Gibbs free energy of denaturation, of the melting temperature and of the temperature of cold denaturation, were all correctly predicted by the model. Combined effects of multiple denaturants, including the effects of pH in the presence of denaturants other than protons, are also predictable by the model.     

Lentinula edodes (shiitake mushroom): An assessment of in vitro anti-atherosclerotic bio-functionality

Mushrooms have been highly regarded as possessing enormous nutritive and medicinal values. In the present study, we evaluated the anti-oxidative and anti-atherosclerotic potential of shiitake mushroom (Lentinula edodes) using its solvent–solvent partitioned fractions that consisted of methanol:dichloromethane (M:DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA) and aqueous residue (AQ). The hexane fraction (1 mg/mL) mostly scavenged (67.38%, IC₅₀ 0.55 mg/mL) the 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical, contained the highest reducing capacity (60.16 mg gallic acid equivalents/g fraction), and most potently inhibited lipid peroxidation (67.07%), low density lipo-protein oxidation and the activity of 3-hydroxy 3-methyl glutaryl co-enzyme A reductase (HMGR). GC–MS analyses of the hexane fraction identified α-tocopherol (vitamin E), oleic acid, linoleic acid, ergosterol and butyric acid as the bio-functional components present in L. edodes. Our findings suggest that L. edodes possesses anti-atherosclerotic bio-functionality that can be applied as functional food-based therapeutics against cardiovascular diseases.