Protein Denaturation

Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley powdery mildew Candidate Secreted Effector Proteins (CSEPs), CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, 16.9 and 17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins including defense-related signaling components through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when co-expressed with Hsp16.9 and Hsp17.5, confirming the ...

The binding of EGTA and EDTA to a-lactalbumin, first demonstrated by Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258,5707-5709) and afterwards regarded as a significant source of error in estimating the binding constant of Ca" to the protein, has been investigated by comparison of the thermal unfolding curves of the protein in the absence and presence of the chelators and also by measuring the NMR spectra of the protein, the chelators, and the protein-chelator mixtures. The unfolding curve in the presence of a large excess of each chelator has been found to be identical to that in the absence of chelator, indicating that there is essentially no interaction between the chelators and a-lactalbumin. The NMR results have also supported this conclusion, and the innocuous character of these chelators as metal-ion buffers in studying the Ca2+-binding properties of a-lactalbumin is demonstrated. In order to re-examine the binding constant for Ca2+ of a-lactalbumin without the aid of chelating metal-ion buffers, the thermal unfolding curve of the protein in the presence of 0.1 mM excess Caz+ but without chelators has been compared with the unfolding curve in the absence of Ca2+ at a constant concentration of Na+ (0.010 or 0.10 M) at pH 7.0. The binding constant of a-lactalbumin can be calculated from the increment of melting temperature caused by the presence of Ca" and from the enthalpy and heat capacity changes in the unfolding. Because Ca2+ binding to the unfolded protein can be neglected under the conditions employed, the binding constant evaluated corresponds to the binding constant to protein that has native structure. The constant obtained is 3-5 X loe M" after corrections for binding of Na+ to the protein and for ionic strength, and this shows excelient agreement with the corresponding value previously estimated (2.9 2 1.0 x lo@ M-'), although the latter value was obtained in the presence of EDTA. The apparent Caz+-binding constant that has been discussed in most previous studies, without taking account of the folding-unfolding equilibrium associated with the binding process, also depends on the concentration of monovalent cations such as Na+, and the present results lead to values of 1.5 x 10' and 8.7 X 10' M-' at 0.01 and 0.1 M Na+, respectively.

The molten globule state is an intermediate between the native and the fully unfolded states of globular proteins and is purported to be an obligatory on-pathway intermediate of protein folding. The molten globule state of ␣-lactalbumin has been best characterized, but two major issues have yet to be clarified. At which stage of the kinetic refolding is the molten globule state stably organized? And what is the major driving force that stabilizes the molten globule state? We address these questions in this paper. We have investigated the refolding kinetics of ␣-lactalbumin using stopped-flow CD and fluorescence, acrylamide quenching and pulsed hydrogen exchange NMR techniques. A burst-phase intermediate was observed to form within 15 ms. The intermediate was characterized by pronounced, hydrogenbonded secondary structure, exposure of hydrophobic surfaces and the absence of tertiary structure. Furthermore, the stability of the secondary structure is the same as that in the equilibrium molten globule state. The burst-phase intermediate in ␣-lactalbumin refolding is identical with the molten globule state. Two different models, the hydrophobic collapse model and the secondary-structure coalescence model, of protein folding are discussed on the basis of the present results. The importance of solventseparated hydrophobic interactions that stabilize the molten globule state is proposed.

We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small‐angle solution X‐ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea‐induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil‐like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X‐ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same struct...

The transition of prion protein from a mainly α-structured isoform (PrPC) to a β sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in α and β conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (S3°C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high β sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native α-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was...

Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric three-stranded β-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-β-sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo , ...

Modern cosmology gives us the largest context within which to understand the emergence of life in the universe. Within this context, the transition from molecular to biological evolution is the most recent in a series of transitions that can be easily described as the result of decreasing temperature. In the big bang model for the evolution of the universe, as the universe expanded and cooled from arbitrarily high temperatures, a universal deterministic cascade of structure appeared. Here we describe how the expansion and decreasing temperature of the universe allowed atoms, molecules, stars, planets and life to form. We discuss the thermal constraints on terrestrial biogenesis and suggest that they apply to the earliest forms of life, not only on Earth, but anywhere in the universe. Cosmology, physics and chemistry are deterministic in the sense that what we learn on Earth will be valid in the most distant parts of the universe. Biological evolution on the other hand seems to be dominated by quirky tinkering and historical contingency. We do not expect elephants or tuataras to have evolved anywhere else in the universe. If life emerged from non-life through a process of molecular evolution, there must be a continuum or a transition between these two paradigms. The first steps in the transition will be inevitable and temperature-induced. The next steps will be less deterministic and the final steps will be as highly contingent and unpredictable as life is today. Here we describe the first thermal constraints of an abiotic deterministic universe and the transition to a less-predictable biotic one. This thermal determinism is similar to the biochemical determinism of "Vital Dust" . deDuve argues that biogenesis and much of what life is, is biochemically inevitable. Here we argue that biogenesis and much of what life is, is thermally inevitable. The very early universe was filled with radiation at arbitrarily high temperature (Fig. ). At ~ 10 -33 seconds after the big bang, the universe cooled enough to allow matter and anti-matter to annihilate, converting rest mass into radiation while leaving a small excess (one part in one billion) of matter. This transition is known as baryogenesis and is the formation of all the matter in the universe. At ~10 -4 seconds after the big bang the

Size exclusion high-performance liquid chromatography analysis was carried out on wheat glutenglycerol blends subjected to different heat treatments. The elution profiles were analyzed in order to follow the solubility loss of protein fractions with specific molecular size. Owing to the known biochemical changes involved during the heat denaturation of gluten, a mechanistic mathematical model was developed, which divided the protein denaturation into two distinct reaction steps: (i) reversible change in protein conformation and (ii) protein precipitation through disulfide bonding between initially SDS-soluble and SDS-insoluble reaction partners. Activation energies of gluten unfolding, refolding, and precipitation were calculated with the Arrhenius law to 53.9 kJ‚mol -1 , 29.5 kJ‚mol -1 , and 172 kJ‚mol -1 , respectively. The rate of protein solubility loss decreased as the crosslinking reaction proceeded, which may be attributed to the formation of a three-dimensional network progressively hindering the reaction. The enhanced susceptibility to aggregation of large molecules was assigned to a risen reaction probability due to their higher number of cysteine residues and to the increased percentage of unfolded and thereby activated proteins as complete protein refolding seemed to be an anticooperative process.

We have compared the behavior of ovotransferrin at the air-solution interface in the presence of a monovalent ion (acetate), or a divalent ion (citrate), the latter being known to induce conformational changes of this protein upon interaction with its iron-binding sites. We have characterised the adsorption layer at the air-water interface in terms of homogeneity, surface concentration excess and rheological properties at pH 4.0. Besides we have investigated the bulk conformation in the presence of the two anions. In the presence of citrate only, interfacial layers display well-defined domains of higher overall surface concentration suggesting multilayers adsorption. Citrate also induces higher helical content and stabilises the protein against thermal denaturation. Hence we propose that these changes are involved in the propensity of ovotransferrin to self-assemble at the air-water interface resulting in thick and heterogeneous interfacial layer.

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive -peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ' SDN loop ' by Ala or Gly significantly decreased the k cat \K m value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate

The mitochondrial membrane-associated carnitine palmitoyltransferase system is a validated target for the treatment of type 2 diabetes mellitus. To further facilitate structure-based drug discovery, we determined the crystal structure of rat CPT-2 (rCPT-2) in complex with the substrate analogue palmitoyl-aminocarnitine at 1.8 A ˚resolution. Biochemical analyses revealed a strong effect of this compound on rCPT-2 activity and stability. Using a computational approach we examined the membrane association of rCPT-2. The protein interacts with the membrane as a functional monomer and the calculations confirm the presence of a membrane association domain that consists of layers of hydrophobic and positively charged residues.

Maintenance of cellular redox homoeostasis forms an important part of the cellular defence mechanism and continued cell viability. Despite extensive studies, the role of the chaperone Hsp104 (heat-shock protein of 102 kDa) in propagation of misfolded protein aggregates in the cell and generation of oxidative stress remains poorly understood. Expression of RNQ1-RFP in Saccharomyces cerevisiae cells led to the generation of the prion form of the protein and increased oxidative stress. In the present study, we show that disruption of Hsp104 in an isogenic yeast strain led to solubilization of RNQ1-RFP. This reduced the oxidative stress generated in the cell. The higher level of oxidative stress in the Hsp104-containing (parental) strain correlated with lower activity of almost all of the intracellular antioxidant enzymes assayed. Surprisingly, this did not correspond with the gene expression analysis data. To compensate for the decrease in protein translation induced by a high level of...

Sso7d from the thermoacidophilic archaebacterium Sulfolobus solfataricus is a small globular protein with a known three-dimensional structure. Inspection of the structure reveals that Phe31 is a member of the aromatic cluster forming the protein hydrophobic core, whereas Trp23 is located on the protein surface and its side chain exposed to the solvent. The thermodynamic consequences of the substitution of these two residues in Sso7d have been investigated by comparing the temperature-induced denaturation of Sso7d with that of three mutants: F31A-Sso7d, F31Y-Sso7d, and W23A-Sso7d. The denaturation processes proved to be reversible for all proteins, and represented well by the two-state N S D transition model in a wide range of pH. All three mutants are less thermally stable than the parent protein; in particular, in the pH range of 5.0-7.0, the F31A substitution leads to a decrease of 24 °C in the denaturation temperature, the F31Y substitution to a decrease of 10 °C, and the W23A substitution to a decrease of 6 °C. A careful thermodynamic analysis of such experimental data is carried out.

Protein glycosylation is a ubiquitous post-translational modification found in all domains of life. Despite their significant complexity in animal systems, glycan structures have crucial biological and physiological roles, from contributions in protein folding and quality control to involvement in a large number of biological recognition events. As a result, they impart an additional level of 'information content' to underlying polypeptide structures. Improvements in analytical methodologies for dissecting glycan structural diversity, along with recent developments in biochemical and genetic approaches for studying glycan biosynthesis and catabolism, have provided a greater understanding of the biological contributions of these complex structures in vertebrates.

Mutation of the glycine 98 residue to arginine in αA-crystallin has been shown to cause presenile cataract in an Indian family. Our earlier study showed that the mutant protein exhibits folding defects that lead to aggregation and inclusion body formation in Escherichia coli. Despite the presence of a normal copy, the pathology is seen in the heterozygous individuals. Formation of mixed oligomers between wild-type and the mutant subunits might be crucial for manifestation of such dominant negative character. We have investigated the role of G98R mutation in αAcrystallin in its structural stability and subunit exchange. G98R αA-crystallin unfolds at lower concentrations of urea compared to wild-type αA-crystallin. The mutant protein is more susceptible to proteolysis than the wild-type protein and transiently populates fragments that are prone to aggregation. Subunit exchange studies using fluorescence resonance energy transfer show that the mutant protein forms mixed oligomers with the wild-type protein. The mutant protein is more susceptible to thermal aggregation, whereas mixed oligomer formation leads to a decreased propensity to aggregate. Coexpression of wild-type αA-crystallin with G98R αA-crystallin in E. coli rescues the mutant αA-crystallin from formation of inclusion bodies. These observations may underlie the molecular basis for the presenile onset, not congenital cataract, in spite of severe folding defect and aggregation of the mutant. Our study shows that the mixed oligomers of wild-type and G98R αA-crystallin exhibit properties dominated by those of the mutant protein in structural aspects, oligomeric size, urea-induced unfolding and, more importantly, the chaperone activity, which may provide the molecular basis for presenile cataract formation in affected individuals.

We introduce coherent infrared emission interferometry as a χ (2) vibrational spectroscopy technique and apply it to studying the initial dynamics upon photoactivation of myoglobin (Mb). By impulsive excitation (using 11-fs pulses) of a Mb crystal, vibrations that couple to the optical excitation are set in motion coherently. Because of the order in the crystal lattice the coherent oscillations of the different proteins in the crystal that are associated with charge motions give rise to a macroscopic burst of directional multi-teraHertz radiation. This radiation can be detected in a phase-sensitive way by heterodyning with a broad-band reference field. In this way both amplitude and phase of the different vibrations can be obtained. We detected radiation in the 1,000–1,500 cm −1 frequency region, which contains modes sensitive to the structure of the heme macrocycle, as well as peripheral protein modes. Both in carbonmonoxy-Mb and aquomet-Mb we observed emission from six modes, whic...

As a physical reaction to injury, infection, or destruction, inflammation is characterized by heat, redness, discomfort, swelling, and abnormal physiological processes. The inflammatory process is a complicated series of relationships among soluble substances and cellular elements that frequently include connective tissue damage. An uncontrolled inflammatory response is the primary cause of a wide range of disorders, including allergies, cardiovascular dysfunctions, metabolic syndrome, cancer, and autoimmune diseases, placing a significant financial burden on individuals and, in turn, on society. Steroids, nonsteroid anti-inflammatory drugs, and immunosuppressants are some examples of common medications for controlling and suppressing inflammatory crises; but they have several side effects, particularly stomach irritation that can cause gastric ulcers. Using natural goods has had a significant positive impact on the development of modern medicine. On such background, plants have historically been an excellent source of medication, and there are several records on the use of herbs in traditional medicine to treat a variety of inflammatory illnesses. However, there has been no scientific attempt to verify these spontaneous uses reported in the literature. Therefore, to conduct more studies and gather more data, scientists need to develop low-cost, straightforward in vitro methodologies to assess the efficacy of natural anti-inflammatory compounds. With this context in mind, this methodology focuses on in vitro anti-inflammatory methodology that can be used to evaluate natural anti-inflammatory substances. They are cost-effective, easy to operate, and dependable with minor alterations.

CORRECTION NOTICE: In the published article titled “In Vitro Anti-Inflammatory Egg Albumin Denaturation Assay: An Enhanced Approach” (Medwin Publishers), an error was identified in the Materials and Methods section. The absorbance measurement wavelength was incorrectly reported as 280 nm due to a typographical oversight. The correct wavelength employed for assessing turbidity associated with protein denaturation is 660 nm, which is appropriate for evaluating the degree of protein aggregation during the assay. This correction does not affect the results or conclusions of the study.

Ca 2؉ -dependent membrane interaction has long been recognized as a general property of the annexin (ANX) family of proteins. More recently, it has become clear that ANXs can also undergo Ca 2؉ -independent membrane interactions at mildly acidic pH. Here we use site-directed spin labeling in combination with circular dichroism and biochemical labeling methods to compare the structure and membrane topography of these two different membrane-bound forms of ANX12. Our results reveal strong similarities between the solution structure and the structure of the Ca 2؉ -dependent membrane-bound form at neutral pH. In contrast, all Ca 2؉independent membrane interactions tested resulted in large scale conformational changes and membrane insertion. Pairs of spin labels that were in close proximity across the interface of different domains of the protein in both the soluble and Ca 2؉ -dependent membrane form were >25 Å apart in the Ca 2؉ -independent membranebound form. Despite these major conformational changes, the overall secondary structure content did not appear to be strongly altered and ANX12 remained largely helical. Thus, Ca 2؉ -independent membrane interaction leads to massive refolding but not unfolding. Refolding did not occur at low pH in the absence of membranes but occurred within a few seconds after phospholipid vesicles were added. The phospholipid composition of the vesicles was an important modulator of Ca 2؉ -independent membrane interaction. For example, cardiolipin-containing vesicles induced Ca 2؉ -independent membrane interaction even at near neutral pH, thereby raising the possibility that lipid composition could induce relatively rapid Ca 2؉ -independent membrane interaction in vivo.

The virulence factor CBP is the most abundant protein secreted by Histoplasma capsulatum, a pathogenic fungus that causes histoplasmosis. Although the biochemical function and pathogenic mechanism of CBP are unknown, quantitative Ca 2+ binding measurements indicate that CBP has a strong affinity for calcium (K D = 6.45 ± 0.4 nM). However, no change in structure was observed upon binding of calcium, prompting a more thorough investigation of the molecular properties of CBP with respect to self-association, secondary structure, and stability. Over a wide range of pH values and salt concentrations, CBP exists predominantly as a stable, noncovalent homodimer in both its calcium-free and-bound states. Solution-state NMR and circular dichroism (CD) measurements indicated that the protein is largely α-helical, and its secondary structure content changes little over the range of pH values encountered physiologically. ESI-MS revealed that the six cysteine residues of CBP are involved in three intramolecular disulfide bonds that help maintain a highly protease resistant structure. Thermally and chemically induced denaturation studies indicated that unfolding of disulfide-intact CBP is reversible and provided quantitative measurements of protein stability. This disulfide-linked, protease resistant, homodimeric α-helical structure of CBP is likely to be advantageous for a virulence factor that must survive the harsh environment within the phagolysosomes of host macrophages.

Fourier transform infrared difference spectroscopy (FTIR) reveals that the Meta II intermediate of the rhodopsin bleaching cascade is structurally distorted relative to rhodopsin. In addition to previously detected alterations in the state of carboxyl groups, a small part of the protein back-bone undergoes a conversion from a-helical to :-type structure. All of these changes partially reverse during Meta II decay. This evidence together with FTIR studies of earlier photointermediates indicates that of the known photointermediates the protein structure of Meta II is the most distorted. It is concluded that light causes rhodopsin to convert into a conformationally distorted form (Meta II), which subsequently refolds into a more rhodopsin-like conformation (opsin).

We develop a machine learning tool useful for predicting the instantaneous dynamical state of sub-monomer features within long linear polymer chains, as well as extracting the dominant macromolecular motions associated with sub-monomer behaviors of interest. We employ the tool to better understand and predict sub-monomer A2 domain unfolding dynamics occurring amidst the dominant large-scale macromolecular motions of the biopolymer von Willebrand Factor (vWF) immersed in flow. Results of coarse-grained Molecular Dynamics (MD) simulations of non-grafted vWF multimers subject to a shearing flow were used as input variables to a Random Forest Algorithm (RFA). twenty unique features characterizing macromolecular conformation information of vWF multimers were used for training the RFA. The corresponding responses classify instantaneous A2 domain state as either folded or unfolded, and were directly taken from coarse-grained MD simulations. three separate RFAs were trained using feature/response data of varying resolution, which provided deep insights into the highly correlated macromolecular dynamics occurring in concert with A2 domain unfolding events. the algorithm is used to analyze results of simulation, but has been developed for use with experimental data as well. von Willebrand Factor (vWF) is a large multimeric protein found in blood plasma. vWF plays an indispensable role in the blood clotting process by initiation of clot formation that stops bleeding due to vessel hemorrhage . vWF is able to sense elevated hydrodynamic force in blood flow at the site of vascular damage and respond by undergoing conformational changes . These changes occur on multiple length scales. First, vWF unravels from a compact globule to an unraveled configuration on a macromolecular length scale, after which a second conformational change may take place on a sub-monomer length scale whereby the A2 domain of vWF is able to unfold if hydrodynamic force is sufficient . This study focuses on the A2 domain of the vWF monomer. Single-molecule pulling experiments have demonstrated that the A2 domain unfolds due to internal tensile forces in the range of 7-15 pN 10 , where larger unfolding forces were observed for faster pulling rates. Thus, under the action of hydrodynamic forces in shearing flows, it is expected that A2 domains unfold when subject to forces in the range reported from single-molecule pulling experiments. The partial or complete unfolding of the A2 domain is required for molecular scission by the enzyme ADAMTS13 , because the vWF cleavage site is deeply buried in the folded A2 domain structure and

Pauling's Paradox and the Release Factor Solution Minato-ku, Tokyo 108-8639 In addition to the recognition of two stop codons each, Japan RF1 and RF2 must discriminate against the 61 sense † Department of Cell and Molecular Biology codons of the genetic code. This negative function may Uppsala University well be the most difficult design feature of termination S-75124 Uppsala of protein synthesis. Linus Pauling argued that the ability Sweden of proteins to discriminate between similar substrates is quite limited. If true this would lead to the apparent paradox that protein synthesis is much more accurate in amino acid selection than the physical chemistry The termination of protein synthesis takes place on the seems to allow. Pauling's "paradox" was finally resolved ribosomes as a response to a stop, rather than a sense, when it was realized that the accuracy of enzymatic codon in the "decoding" site (A site). Polypeptide reselections can be enhanced by energy-driven, multiple lease factors (RFs) play an essential role in this process. step "proofreading" of substrates (Kurland et al., 1996, Although the termination process and the RF activity and references therein). The mimicry hypothesis sugwere discovered in the late 1960s (Goldstein and Caskey, 1970, and references therein), much of the gests that class II RFs share structural and functional mechanisms remained obscure. After three decades of properties with elongation factor EF-Tu (eEF-1␣) or EF-G investigation, we now know that two classes of RFs are (Nakamura et al., 1996, and references therein). At face required for translation termination in prokaryotes and value mimicry would imply that class II RFs enhance the eukaryotes: a class I factor, codon-specific RFs (RF1 for speed of peptide release since EF-Tu accelerates the UAG/UAA and RF2 for UGA/UAA in prokaryotes; eRF1 in rate of peptidyl transfer to aminoacyl-tRNA by orders eukaryotes), and a class II factor, nonspecific RFs (RF3 of magnitude. It would also imply that the very tight in prokaryotes; eRF3 in eukaryotes) that bind GTP or discrimination against premature termination at sense GDP and stimulate class I RF activity (reviewed by Nakacodons observed in bacteria depends on proofreading mura et al., 1996). In contrast, eukaryotic eRF1s normally

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Stra ¨ussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues ϳ82-146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82-146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82-146 small oligomers (1-5-mers, flowing species) onto soluble prefibrillar PrP82-146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82-146 flowed onto the scrambled sequence of PrP82-146 or onto prefibrillar A␤42 aggregates. As previously found with A␤40, SPR data could be adequately fitted by equations modeling the "dock-and-lock" mechanism, in which the "locking" step is due to sequential conformational changes, each increasing the affinity of the monomer for the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82-146 than with A␤40. Such differences suggest that PrP82-146 has a greater propensity to polymerize and greater stability of the aggregates. Prion diseases are transmissible neurodegenerative disorders characterized by the transition of the prion protein (PrP) 3 from an ␣-helix-rich soluble isoform (PrP C ) into disease-specific insoluble species (PrP Sc ) with a prevalent ␤-sheet structure (1, 2). A major feature of PrP Sc is

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-β-d-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Ce manuscrit propose une modélisation rigoureuse en 40 chapitres de la genèse moléculaire du vivant, depuis la chimie abiotique jusqu’à l’apparition de protocellules fonctionnelles. L’approche repose sur cinq piliers — synthèse, polymérisation, compartimentation, catalyse primitive et réplication — et mobilise les interactions physico-chimiques, les cycles redox et les gradients environnementaux comme vecteurs de structuration. Ce cadre constitue la première phase d’un développement approfondi en 174 chapitres, destiné à modéliser de façon exhaustive la transition moléculaire vers les systèmes vivants.
This manuscript presents a rigorous 40-chapter mechanistic modeling framework of molecular genesis, tracing the evolution from abiotic chemistry to functional protocellular systems. Structured around five fundamental pillars—molecular synthesis, polymerization, compartmentalization, primitive catalysis, and replicability—the approach emphasizes the role of physicochemical interactions, redox cycles, and environmental gradients in molecular complexity. This framework constitutes the initial phase of an extensive 174-chapter development aimed at exhaustively modeling the molecular transition toward living systems.

All-atom molecular dynamics simulations of proteins in solvent are now able to realistically map the protein-unfolding pathway. The agreement with experiments probing both folding and unfolding suggests that these simulated unfolding events also shed light on folding. The simulations have produced detailed models of protein folding transition, intermediate, and denatured states that are in both qualitative and quantitative agreement with experiment. The various studies presented here highlight how such simulations both complement and extend experiment.

Proteasome inhibition represents an important anticancer strategy. Here, we studied the mechanisms at the basis of the pro-apoptotic activity of the standardized decoction of Hemidesmus indicus, a plant evoking a complex anticancer activity, and explored its inhibition of proteasome activity in human leukemia cells. Additionally, we preliminary tested the cytotoxicity of some H. indicus’s phytochemicals on leukemia cells and their intestinal absorption on a human intestinal epithelium model consisting of a monolayer of differentiated Caco2 cells. We observed a potent antileukemic effect for H. indicus, imputable to the modulation of different critical targets at protein and mRNA levels and the reduction of the 26S proteasome expression. We found that some phytomarkers of H. indicus decoction passed through the enterocyte monolayer. Overall, our study supports the pharmacological potential of H. indicus, which can represent an interesting botanical drug in the oncological area.

Apolipoprotein E (apoE) enters the plasma as a component of discoidal HDL and is subsequently incorporated into spherical HDL, most of which contain apoE as the sole apolipoprotein. This study investigates the regulation, origins, and structure of spherical, apoE-containing HDLs and their remodeling by cholesteryl ester transfer protein (CETP). When the ability of discoidal reconstituted high density lipoprotein (rHDL) containing apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein to act as substrates for LCAT were compared with that of discoidal rHDL containing apoA-I [(A-I)rHDL], the rate of cholesterol esterification was (A-I)rHDL .. (E2)rHDL z (E3)rHDL . (E4)rHDL. LCAT also had a higher affinity for discoidal (A-I)rHDL than for the apoEcontaining rHDL. When the discoidal rHDLs were incubated with LCAT and LDL, the resulting spherical (E2)rHDL, (E3)rHDL, and (E4)rHDL were larger than, and structurally distinct from, spherical (A-I)rHDL. Incubation of the apoE-containing spherical rHDL with CETP and IntralipidT generated large fusion products without the dissociation of apoE, whereas the spherical (A-I)rHDLs were remodeled into small particles with the formation of lipid-poor apoA-I. In conclusion, i) apoE activates LCAT less efficiently than apoA-I; ii) apoE-containing spherical rHDLs are structurally distinct from spherical (A-I)rHDL; and iii) the CETPmediated remodeling of apoE-containing spherical rHDL differs from that of spherical (A-I)rHDL.-Rye, K-A., R.

The NADP'-dependent glutamate dehydrogenase from Thernu~coccus litoralis has been crystallized by the hangingdrop method of vapour diffusion using an ammonium sulfate and PEG mixture as the precipitant. The crystals belong to the monoclinic system and are in space group,(~2 with unit-cell dimensions a = 142.7, h=202.0, c = 125.8A with [~= 113.1 :~ with a hexamer in the asymmetric unit. T. Litoralis, a hyperthermophilic organism, belongs to the family of Archaea and has a maximum growth temperature of about 370 K. The glutamate dehydrogenase isolated from this organism has a halt-life of 2 h at 373 K and a comparison of this structure with that of other GluDtI's from hyperthermophilic organisms and from mesophiles will contribute to an understanding of the molecular mechanisms which underlie thermostability.

In laser spray, the tip of an electrospray capillary is irradiated with a continuous CO 2 laser beam. Here, we report results from a modified laser spray method that employs a relatively low laser irradiance level. With a laser power of ϳ2 W and a focal spot size (ϳ0.3 mm), which covered the entire front surface of the electrospray capillary, the irradiance was ϳ3 ϫ 10 3 W/ cm 2 . This resulted in a quiescent and smooth vaporization of aqueous solutions. This "evaporation-mode" laser spray method yielded the best results so far obtained in our laboratory with laser-irradiated electrospray, producing higher and more stable signals. The method was applied to the analysis of aqueous solutions of lysozyme and myoglobin. Mass spectra were obtained as a function of laser power from 0 W (electrospray) to ϳ2 W. The spray generated at the tip of the stainless steel capillary was observed with a CCD camera. With increase of laser power, the droplets in the spray became finer and the Taylor cone became progressively smaller. The strongest ion signals were recorded when the sample solution protruded only slightly from the tip of the capillary. A broadening of the lysozyme charge-state distribution, attributable to protein unfolding, was observed with a laser power of 2 W. No denaturation of myoglobin took place up to a laser power of 1.6 W. However, a sudden onset of denaturation was observed at 1.8 W as a broadening of the myoglobin charge distribution and the appearance of apo-myoglobin peaks. These findings demonstrate that laser spray is capable of dissociating the noncovalent complexes selectively without breaking covalent bonds. (

We consider here the possibility of shaping the high-frequency acoustic propagation in a liquid upon directional confinement. This hypothesis is investigated by Inelastic X-ray Scattering measurements on water confined in aligned multi-walled carbon nanotubes, in which the momentum transfer was either parallel or orthogonal to the confinement axis. The comparison between the spectra measured in these two scattering geometries highlights the anisotropic nature of the dynamic response of directionally confined water, thus potentially inspiring new pathways to shape high-frequency sound propagation in isotropic systems. Finally, a Bayesian analysis of measured spectra unravels close similarities in the phonon dispersions of multiwalled nanotubes and graphite.

The gene encoding a cellobiohydrolase (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, sub-cloned and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kD. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module (CBM). Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55°C with methylumbelliferryl-cellobioside (MUC) as the substrate and retained 85% of its activity following 24 h incubation at 50°C. Despite the lack of activity towards microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy suggested a reduction of lignin and crystalline cellulose ...

The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 °C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate isomerase (tPRAI) from the hyperthermophile Thermotoga maritima is at its optimum growth temperature of 80 °C, and how its thermolabile substrate, N‐(5′‐phosphoribosyl)‐anthranilate (PRA), is protected from rapid decomposition. To this end, the trpF gene of T. maritima was expressed heterologously in Escherichia coli and tPRAI was purified. In contrast to most PRAIs from mesophiles, which are monomers with the eightfold βα (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 °C, by acidification to pH 3.2, and by proteases in the presence and absence of detergents. tPRAI is about 35‐fold more active at its physiologic temperature than i...

The authors showed temperature-independent effects of microwave and far-IR electric fields on the secondary structure of proteins. However, the requisite extreme field strengths are unachievable in the environment or laboratory. Because protein bond effects are amplitude dependent, exposures to subthreshold field strengths would have no effect regardless of exposure duration.

The occurrence of age-related modifications in functional and structural properties of several enzymes has been documented; however, the molecular basis of this phenomenon is still mostly unexplained. In the present work a comparative study of phosphoglycerate kinase preparations isolated from hearts of young and old rats was undertaken. Marked age-related effects were revealed in the heat-inactivation kinetics of the enzyme, similar to the ones previously found in purified muscle phosphog|ycerate kinase. In view of the previously reported failure of immunotitration to distinguish between phosphog|ycerate kinase forms in cru~e heart extracts from young and old rats, it appears likely that the modifications in old rat heart phosphoglycerate kinase are in a domain which is not involved in antibody binding, and may be localized in the interior of the enzyme. These age-related modifications were completely relieved by extensive unfolding of the enzyme in 2 M guanidine hydrochloride, followed by enzyme reactivation upon dilution of the denaturant. The refolding products of young and old enzymes displayed identical heat-inactivation kinetics as native young phosphoglycerate kinase. It is concluded that the age-related alterations in rat cardiac phosphoglycerate kinase, like those found in the muscle enzyme, are purely conformational and hence develop postsynthetically.

) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT 4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55°C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.

Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state with more compact structures, and therefore, a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found between experimental and calculated folding rates based on free energy barrier heights using the same diffusion coefficient for every protein.

Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state with more compact structures, and therefore, a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found between experimental and calculated folding rates based on free energy barrier heights using the same diffusion coefficient for every protein.

Equilibrium measurements. Equilibrium fluorescence was measured with a Spex Fluorlog spectrofluorimeter using 20 μM protein samples excited at 284nm. Absolute quantum yields were calculated by integrating the emission spectra from 300 nm to 450 nm and scaling to N-acetyltryptophanamide as a reference assuming a quantum yield of 0.14 at 293K. A Jasco 720 spectropolarimeter was utilized for circular dichroism (CD) measurements (protein concentration of 10 μM in a 1 cm quartz cuvette).

BACKGROUND: Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies, where the protein deposition occurs, and in the biotechnology field like the food technology where many processes in food manufacturing are based on thermal treatments. OBJECTIVE: The influence of Cu 2+ or Zn 2+ ions on the thermal aggregation process of Bovine β-lactoglobulin (BLG) and Bovine Serum Albumin (BSA), two protein models, was studied with the aim of delineating the role of these ions in the protein aggregation kinetics and to clarify the related molecular mechanisms. METHODS: The protein structure changes were monitored by Raman spectroscopy, whereas the aggregate growth was followed by Dynamic Light Scattering measurements. RESULTS: Both metal ions are able to favour the BLG aggregation, whereas only Zn 2+ ions have a promoter effect on the thermal aggregation of BSA. The reason of this different behaviour is that the BLG aggregation evolution is manly affected by the redistribution of charges, whereas that of BSA by the metal coordination binding which depends on metal. CONCLUSIONS: Raman spectroscopy, combined with dynamic light scattering experiments, was very useful in identifying the role played by Cu 2+ and Zn 2+ on the aggregation pathways of BLG and BSA. The results provide evidence for the role of histidine residues both in the redistribution of charges and in the two modes of metal binding that take place in BLG-and BSA-containing systems, respectively.

In the present study we have investigated deleterious mutants in the uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutants found in CEP patients have been cloned, expressed, and purified. Their enzymatic activities have been measured relative to wild-type UROIIIS activity. All mutants retain measurable activity, consistent with the recessive character of the disease. Most of the mutants with a significant decrease in activity involve residues likely associated in binding. However, other mutants are fully active, indicating that different mechanisms may contribute to enzyme missfunction. UROIIIS is a thermolabile enzyme undergoing irreversible denaturation. The unfolding kinetics of wild-type UROIIIS and the suite of mutants have been monitored by circular dichroism. This analysis allowed the identification of a helical region in the molecule, essential to retain the kinetic stability of the folded conformation. C73R is found in onethird of CEP patients, and Cys73 is part of this helix. The integrated analysis of the enzymatic activity and kinetic stability data is used to gain insight in the relationship between defects in UROIIIS sequence and CEP.