Protein Stability

We have previously reported the identification of a small, basic and cysteine-rich antifungal peptide (AcAFP) secreted by Aspergillus clavatus and shown its ability to prevent growth of various human-and plant-pathogenic filamentous fungi. In this study, we sought to determine the physiological/microbiological requirements to enhance the AcAFP production and the conditions influencing its stability. The maximum of AcAFP production was obtained when A. clavatus was grown on 2% glycerol as sole carbon source and 100 mM NaCl. The AcAFP expression was shown to be influenced by pH, being suppressed under acidic (pH 5) and strongly induced under alkaline conditions. The activity of the purified AcAFP was not affected by temperature; it loosed approximately 20% of its activity after 3 h at 100°C and was efficient through a large pH range (pH 5-12) with an optimum at pH 8. AcAFP activity decreased at high ionic strength and in the presence of 10 mM of divalent cations (Mn 2+ , Fe 2+ and Ca 2+ ).

Introduction: Guidelines recommend storing tuberculin purified protein derivative (PPD) refrigerated. However, especially in developing countries, maintaining the product refrigerated under field conditions can be difficult, limiting its use. Here we determine the effect of prolonged exposure to high temperatures on the potency of human, bovine and avian tuberculin PPD. Methodology: Human, bovine and avian tuberculin PPD were stored for several weeks exposed to temperatures ranging from 37º to 100ºC. The potency was evaluated in vivo, in sensitized or naturally infected animals. Results: Most test situations didn't affect the biological activity of the tuberculin PPDs and only very long and extreme incubations (several days at 100°C) compromised the potency. Conclusions: Tuberculin PPD is very stable and can be stored or transported for long periods without refrigeration. 

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, we studied wild type bR and especially the triple mutant bR, 3Glu [E9Q/E194Q/E204Q], in combination with wide gap semiconductor TiO 2 for their suitability as efficient light harvester in solar cell. Our differential scanning calorimetry data show thermal robustness of bR wild type and 3Glu mutant, which make them good candidates as photosensitizer in solar cells. Molecular modeling indicates that binding of bR to the exposed oxygen atoms of anatase TiO 2 is favorable for electron transfer and directed by local, small distance interactions. A solar cell, based on bR wild type and bR triple mutant immobilized on nanocrystalline TiO 2 film was successfully constructed. The photocurrent density-photo voltage (J-V ) characteristics of bio-sensitized solar cell (BSSC), based on the wild type bR and 3Glu mutant adsorbed on nanocrystalline TiO 2 film electrode were measured. The results show that the 3Glu mutant displays better photoelectric performance compared to the wild type bR, giving a short-circuit photocurrent density (J SC ) of 0.09 mA/cm 2 and the open-circuit photovoltage (V OC ) 0.35 V, under an illumination intensity of 40 mW/cm 2 .

In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 628 8 8 8 8C (178 8 8 8 8 higher in comparison with the wt enzyme). Keywords: Moloney Murine leukemia virus reverse transcriptase/processive reverse transcriptase/thermostable reverse transcriptase

Pathogen recognition by the plant immune system is governed by structurally related, polymorphic products of disease resistance (R) genes. RAR1 and/or SGT1b mediate the function of many R proteins. RAR1 controls preactivation R protein accumulation by an unknown mechanism. We demonstrate that Arabidopsis SGT1b has two distinct, genetically separable functions in the plant immune system: SGT1b antagonizes RAR1 to negatively regulate R protein accumulation before infection, and SGT1b has a RAR1-independent function that regulates programmed cell death during infection. The balanced activities of RAR1 and SGT1, in concert with cytosolic HSP90, modulate preactivation R protein accumulation and signaling competence.

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at À80°C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assemblycompetent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.

Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45-47°C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.

Upon the removal of water, proteins undergo a major, reversible rearrangement of their secondary structure, as revealed by FTIR spectroscopy. We have found herein that for recombinant human albumin (rHA) the extent of this structural change does not depend significantly either on the composition of the aqueous solution prior to lyophilization (protein concentration, pH, and the presence of excipients such as dextran or NaCI) or on the mode of dehydration (lyophilization, spray drying, or rotary evaporation), even though these factors profoundly affect rHA's solid-state stability against moisture-induced aggregation. In all cases, the a-helix content of rHA drops from 58% in solution to 25-35% in the dehydrated state, the /3-sheet content rises from 0 to 10-20%, and unordered structures increase from 40% to 50-60%. We have also investigated another model protein, hen egg-white lysozyme, and confirmed that it too undergoes a significant alteration of the secondary structure upon lyophilization. The extent of this structural reorganization has been found to be insensitive to the pH of the aqueous solution prior to lyophilization from pH 1.9 to 5.1, even though the thermal transition temperature (T m) in aqueous solution over this range varies by 30°C.

Tumor growth, progression, and response to the hypoxic tumor microenvironment involve the action of hypoxia-inducible transcription factors, HIF1 and HIF2. HIF is a heterodimeric transcription factor containing an inducible HIFα subunit and a constitutively expressed HIFβ subunit. The signaling pathways operational in macrophages regulating hypoxia-induced HIFα stabilization remain the subject of intense investigation. Here, it was discovered that the PTEN/PI3K/AKT signaling axis controls hypoxia-induced HIF1α (HIF1A) and HIF2α (EPAS1) stability in macrophages. Using genetic mouse models and pan-PI3K as well as isoform-specific inhibitors, inhibition of the PI3K/AKT pathway blocked the accumulation of HIFα protein and its primary transcriptional target VEGF in response to hypoxia. Moreover, blocking the PI3K/AKT signaling axis promoted the hypoxic degradation of HIFα via the 26S proteasome. Mechanistically, a macrophage-dominant PI3K isoform (p110γ) directed tumor growth, angiogenes...

How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leadin...

Thermodynamic parameters were determined for the dissociation of beta-lactoglobulin(b-Lg) at temperatures from À15 to 858C. The effect of temperature on K d (equilibrium constant for dimer monomer dissociation) was described by a secondorder Van't Hoff equation (ln K d AT À2 BT À1 C) or Gibbs±Helmholtz equation. The Gibbs free energy (DG), enthalpy (DH), entropy (DS) and thermal capacity (DC p ) for b-Lg dissociation were evaluated. At 258C standard temperature thermodynamic parameters were DG 0 24.8 (AE0.35) kJ mol À1 , DH 0 57 (AE13) kJ mol À1 , DS 0 92 (AE30) J mol À1 K À1 and DC p 2383 J mol À1 K À1 at pH 2.6. For b-Lg dissociation at pH 7, DG 0 28.6 (AE2.7) kJ mol À1 , DH 0 107.5 (AE6.3) kJ mol À1 , DS 0 265.7 (AE39) J mol À1 K À1 and DC p 2383 J mol À1 K À1 . Simulated temperature±dissociation pro®les of b-Lg show that the fraction of dissociated protein increases with increasing temperature, decreasing pH and with decreasing protein concentration. #

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic control...

Several cytokines have been involved in the diagnosis and prognosis for the pathogenesis and severity of chronic hepatitis B (CHB) such as cluster of differentiation 163 (CD163), neutrophil gelatinase-associated lipocalin (NGAL), high-mobility group box 1 (HMGB1) and macrophage inflammatory protein-2 (MIP-2). Nevertheless, the stability and reliability of these cytokines can be greatly influenced by handling and storage processes. In this study, potential utility of serum samples of a CHB cohort was evaluated to investigate several processes that might impact cytokine profiles such as temperature, storage time and number of freeze-thaw cycles. Blood samples collected from 100 patients with CHB were separated immediately and divided into two groups. In one group, samples (n=50) stored at -80°C were subject to 1-3 freeze-thaw cycles. In the other group, samples (n=50) were stored at 4°C and 25°C for 3 h, 9 h, 24 h, 48 h, 72 h, and 7 d time points, respectively. To assess the influence...

Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 ω3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.Metabolic engineering is focus in the production of a huge diversity of polyunsaturated fatty acids with an economical interest. In this study, we characterized factors contributing to improve the activity of sunflower plastidial ω3-desaturase, HaFAD7, expressed in yeast.

Proteins are marginally stable, and the folding/unfolding equilibrium of proteins in aqueous solution can easily be altered by the addition of small organic molecules known as cosolvents. Cosolvents that shift the equilibrium toward the unfolded ensemble are termed denaturants, whereas those that favor the folded ensemble are known as protecting osmolytes. Urea is a widely used denaturant in protein folding studies, and the molecular mechanism of its action has been vigorously debated in the literature. Here we review recent experimental as well as computational studies that show an emerging consensus in this problem. Urea has been shown to denature proteins through a direct mechanism, by interacting favorably with the peptide backbone as well as the amino acid side chains. In contrast, the molecular mechanism by which the naturally occurring protecting osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins is not clear. Recent studies have established the strong interaction of TMAO with water. Detailed molecular simulations, when used with force fields that incorporate these interactions, can provide insight into this problem. We present the development of a model for TMAO that is consistent with experimental observations and that provides physical insight into the role of cosolvent-cosolvent interaction in determining its preferential interaction with proteins. 273 Annu. Rev. Phys. Chem. 2013.64:273-293. Downloaded from www.annualreviews.org by RENSSELAER POLYTECHNIC INSTITUTE on 04/05/13. For personal use only. Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search Further ANNUAL REVIEWS 274 Canchi · García Annu. Rev. Phys. Chem. 2013.64:273-293. Downloaded from www.annualreviews.org by RENSSELAER POLYTECHNIC INSTITUTE on 04/05/13. For personal use only. Cosolvent: small, neutral organic molecules that can modulate protein stability van't Hoff enthalpy: enthalpy obtained by analysis of the temperature dependence of the equilibrium constant TMAO: trimethylamine N-oxide Molecular dynamics (MD): computational technique to study the microscopic motions of atoms and molecules; molecular trajectories are obtained by numerically integrating Newton's equations of motion Linear extrapolation model (LEM): used to model the dependence of protein stability on cosolvent concentration

ENCoM is a coarse-grained normal mode analysis method recently introduced that unlike previous such methods is unique in that it accounts for the nature of amino acids. The inclusion of this layer of information was shown to improve conformational space sampling and apply for the first time a coarse-grained normal mode analysis method to predict the effect of single point mutations on protein dynamics and thermostability resulting from vibrational entropy changes. Here we present a web server that allows non-technical users to have access to ENCoM calculations to predict the effect of mutations on thermostability and dynamics as well as to generate geometrically realistic conformational ensembles. The server is accessible at: http://bcb.med.usherbrooke.ca/encom.

The effect of Triton X-114 on the physicochemical properties of a single-chain antibody fragment (scFv) has been studied. According to the far UV circular dichroism spectroscopy, the secondary structure of the recombinant antibody was not significantly affected by the presence of Triton. From the antibody tertiary structure analysis, it was found that the surfactant could be located around the tryptophan molecules accessible to the solvent, diminishing the polarity of its environment but maintaining most of the protein structure integrity. However, in certain conditions of high temperature and high concentration of denaturant molecules, the presence of TX could compromise the antibody fragment stability. These results represent a previous step in designing scFv purification protocols and should be considered prior to developing scFv liquid-liquid extraction procedures.

In places previously regarded as incapable of sustaining life, such as Antarctic ice fields and volcanic pools, organisms are now being identified for whom these extreme environments are home, sweet home. In most cases, adaptation to such extreme environments has not required completely new molecular machinery; in fact, many 'extremophilic' enzymes are similar to their counterparts from the 'mesophilic' environments we find more hospitable. For example, enzymes that function at very different temperatures can have nearly superimposable three-dimensional structures . Sequence comparisons indicate that these enzymes are derived from a common ancestral enzyme and have accumulated mutations that allow them to adapt over millions of years.

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

Protein stabilized, water soluble gold nanoparticles are essential for biomedicines and biotechnology. Bovine Serum Albumin (BSA) is one of the most abundant proteins. It has a remarkable ability of binding and transporting materials across cell membrane that makes it ideal for drug delivery and very useful for pharmaceutical industry. Herein, we explored the direct synthesis of BSA stabilized gold nanoparticles via a simple photochemical process. BSA stabilized gold nanoparticles are synthesized in one step, using Irgacure (I-2959) as photoinitiator. UV radiation facilitates the easy one step synthesis of protein stabilized gold nanoparticles without any denaturation of the protein, during the process. Polyacrylamide Gel (PAGE) shows that there is no difference in the bands height and mobility of native BSA and UV irradiated BSA. PAGE results are further confirmed by fluorescence spectroscopy of native and UV irradiated BSA. BSA/PEG (polyethylene glycol) mixed monolayer stabilized gold nanoparticles have also been prepared in one step using the photochemical process. Different ratios of PEG to BSA are used to evaluate the particle size and the size distribution of gold nanoparticles. Transmission electron microscope (TEM) and dynamic light scattering (DLS) confirm the presence of nearly monodisperse mixed layer stabilized gold nanoparticles.

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates (benzoylarginine p-nitroanilide) and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was supported on highly activated glyoxyl agarose following a two-step immobilization strategy: a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pKa amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed, and b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pKa amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a non-stabilized derivative prepared on CNBr. In fact, all the proteins contained in a cheese whey extract had been completely proteolyzed after 6 hours at pH 9 and 50°C, as judged by SDS-PAGE. Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the 3D structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and 5 lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high molecular weight proteins.

Purpose To investigate the cause of unexpected and erratic increase in aggregation during long-term storage of an IgG2 monoclonal antibody in a trehalose formulation at −20°C. Methods Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XP-V patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

Mechanical properties of starch-based supercritical fluid extrusion (SCFX) extrudates were correlated to their structure. Thermosetting ingredients were added to the feed and the extrudate was dried to set the expanded structure. Compared to whey protein concentrate with 34% protein (WPC-34), addition of egg white (EW) gave a softer skin and a fragile but well formed cellular structure. For drying between 70 and 100°C , the overall structure was more homogenous for EW added. A higher modulus was associated with a larger number average of sides in a cell face and higher percentage of closed cells. Ashby's model (1983) for non-food cellular solids gave a good fit to our data for relating relative compressive modulus to relative bulk density. A linear regression model indicated that bulk density alone was a good predictor of mechanical strength.

L’isoforme M2 du pyruvate kinase (PKM2) est responsable de la conversion du phosphoénolpyruvate (PEP) en pyruvate. Cette étape est la dernière étape de catalyse dans le processus de glycolyse puis joue un rôle essentiel dans la fermentation lactique effectuée chez les cellules anaérobiques, notamment les cellules myocardiques. De plus, le pyruvate kinase joue un rôle important dans le métabolisme des cellules cancéreuses. Au cours de cette expérience, nous caractérisent une mutante (K422R) de la PKM2 à l’aide de dosages enzymatiques des deux formes de l’enzyme en présence de certains effecteurs étant déjà identifiés comme régulateurs allostérique de la PKM2. La mutation a été confirmée à l’aide de séquençage et alignement de séquence avec la séquence protéique de la PKM2, retrouvée sur NCBI. Nos résultats démontrent que la mutation K422R favorise l’état T tétramérique et inactive de la PKM2 à cause de formation de liens stabilisantes au sein de la structure globulaire. La PKM2, sous sa forme active, existe plutôt sous l’état R tétramérique (3). Notre étude met en évidence une façon efficace de réguler l’activité enzymatique de la PKM2 à travers un changement dans la structure cristalline du type sauvage. Cette information s’avère utile pour des études dans le traitement du cancer qui vise à inhiber la fonction de la PKM2.

The combination of deuterium–hydrogen exchange (DHX) and mass spectrometry (MS) can be used for studying a high pressure denaturation (HPD) of proteins. Herein we present the results of investigations of the influence of glycation on the HPD of ubiquitin. Application of various values of pressure causes different degrees of protein unfolding, resulting in molecules with a different number of protons available for exchange with deuterons. The dependence of this number on pressure gives information on the denaturation state of a protein. On the basis of the obtained results we can conclude that increasing number of fructosamine moieties in ubiquitin decreases the pressure required for its denaturation. It suggests that glycation moderately decreases the protein stability. The present study is the first example of application of hydrogen–deuterium exchange as a method of investigating the influence of posttranslational modification of protein on the HPD.

Freezing is widely used during the manufacturing process of protein-based therapeutics, but it may result in undesired loss of biological activity. Many variables come into play during freezing that could adversely affect protein stability, creating a complex landscape of interrelated effects. The current approach to the selection of freezing conditions is however nonsystematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on protein stability. Two opposite scenarios are identified, suggesting that the ice-water interface is the dominant cause of denaturation for proteins with high bulk stability, while the duration of the freezing process itself is the key parameter to be controlled for proteins that are susceptible to cold denaturation. Experimental data for lactate dehydrogenase and myoglobin as model proteins support the model results, with a slow freezing rate being optimal for lactate dehydrogenase and the opposite being true for myoglobin. A possible application of the calculated design space to the freezing and freeze-drying of biopharmaceuticals is finally described, and some considerations on process efficiency are discussed as well.

Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) are rare lymphoproliferative cancer types. Although most HL patients can be cured by chemo-and radio-therapy, 4-50% of patients relapse and have a poor prognosis. The need for improved therapeutic options for patients with relapsed or refractory disease has been addressed by CD30-specific antibody-based immunotherapeutics. However, available CD30-specific monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) or chimeric immunotoxins suffer from the requirement of a functional host immunity, undesirable immune reactions or heterogeneity and instability, respectively. Here, we present a new fusion protein comprised of the CD30-specific antibody single-chain fragment Ki4(scFv) and the human pro-apoptotic effector protein, microtubule-associated protein tau (MAPT). Ki4(scFv)-MAP selectively induced apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner. Tubulin polymerization assays confirmed that Ki4(scFv)-MAP stabilizes microtubules, suggesting a mechanism for its pro-apoptotic action. Dosefinding experiments proved that Ki4(scFv)-MAP is well tolerated in mice compared to the previously reported Ki4(scFv)-ETA'. Ki4(scFv)-MAP significantly inhibited growth of subcutaneous L540cy xenograft tumours in mice. Our data present a novel approach for the treatment of CD30 + lymphomas, combining the binding specificity of a target-specific antibody fragment with the selective cytotoxicity of MAPT towards proliferating lymphoma cells.

chapter 2 18 ABSTRACT Therapeutic proteins have become essential in the treatment of many diseases. Their formulation in dry form is often required to improve their stability. Traditional freeze drying or spray drying processes are often harmful to labile proteins, and could be replaced by supercritical fluid (SCF) drying to produce particles with defined physicochemical characteristics in a mild single step. A survey of the current SCF drying processes for proteins is presented to give insight into the effect of SCF drying on protein stability and to identify issues that need further investigation. Methods used for drying aqueous and organic protein solutions are described. In particular, effects of process and formulation parameters on particle formation and protein stability are discussed. Although SCF methodology for drying proteins is still in its infancy, it can provide a serious alternative to existing drying methods for stabilizing proteins.

The thermal stability of b-lactoglobulin (b-Lg) dimer was reassessed based on a three-state denaturation process involving dissociation and unfolding (dimer O monomer O unfolded state). The stabilisation Gibbs free energy change for b-Lg dissociation unfolding (DG O DCU ) was 57.6 kJ mol 21 compared with an estimated 14 kJ mol 21 with b-Lg monomer as the reference native state. The standard enthalpy (DH O ) and entropy (Delta;S O ) change for heat denaturing b-Lg dimer are reported. The new stability parameters are discussed in terms of protein stability function relations. q

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmolykg at 2900 m). Other deep-sea animals had high levels of (1) scyllo-inositol in echinoderms, gastropods, and polychaetes, (2) that polyol plus b-alanine and betaine in octopods, (3) hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4) a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica. ᮊ

Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of: (i) pepsin, (ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of ␤-lactoglobulin (␤-Lg), and especially that of the peptide ␤-Lg f125-135, could have implications for the immunogenic action of ␤-Lg in the insurgence of cow's milk allergy.

There have been many attempts to understand the mechanisms that determine the activity and stability of enzymes at extreme temperatures. Previous studies often compared enzymes from mesophiles and thermophiles and speculated that the observed differences were factors involved in adaptation to a certain temperature range. Most of these studies concluded that gross structural changes such as the quantity of certain amino acids or the total number of hydrogen bonds are responsible for the adaptation of optimal activity to temperature extremes. However, these studies have compared similar enzymes from distantly related organisms leaving one to wonder if the differences noted are from an adaptation to temperature or merely the result of genetic drift.
In this dissertation, I present an analysis of the cold-active beta-galactosidase (BgaS) from the Arthrobacter sp. SB. A study of the primary sequence and modeled structure of BgaS showed that many of the proposed adaptations for optimal activity in the cold did not hold true for this enzyme. Consequently, I decided to alter the BgaS enzyme to determine the contributions of specific amino acids to activity and stability at low temperatures.
I first examined the area of BgaS that aligned with the domain five mobile loop of the LacZ beta-galactosidase of Escherichia coli. In LacZ this area (residues 794-803) aids the binding of substrate and alterations at residue 794 increased the catalytic efficiency of the enzyme with lactose. However, when similar mutations were made in bgaS, they caused either a complete loss or a decrease of activity showing that although this area is also important for BgaS function the alterations affect the enzymes differently.
To further explore the low temperature activity of BgaS, I screened for second site revertants of a null mutant resulting from a G803D change. Restoration of activity was accomplished with the addition of only two mutations (E229D and V405A). Separation of these two mutations into a wild type background yielded an enzyme with a three fold increase in catalytic efficiency with little effect on the thermostability. This shows that small and subtle changes to the enzyme can further increase the activity at low temperatures.
I also explored the thermostability of BgaS through directed mutagenesis and directed evolution studies. The rational mutagenesis targeted the C-terminal portion of BgaS, an area in LacZ known to affect thermostability. From this study, I discovered that a single cysteine to glycine or cysteine to serine mutation resulted in an increase in thermal optimum for BgaS and in another closely related enzyme. The directed evolution study primarily targeted the active site in an attempt to create a more active version of BgaS. One mutant obtained from this screen, with a single alteration in amino acid sequence, created an enzyme with more activity and a 3.3 fold increase in the time it remained active at 30C, compared to BgaS.
Through the combination of mutational analysis and biochemical characterization, I have shown that the introduction of a limited number of amino acid changes are sufficient to alter the activity and/or thermal properties of an enzyme whereas previous studies have suggested that multiple alterations would be required. I have also increased the thermal optimum of three closely related enzymes by altering one amino acid. Considering the large size of the beta-galactosidase subunit, the finding that one change in the C-terminal 25 residues has any effect on the enzyme, not to mention an up to 20C increase in temperature optimum, is quite interesting. Taken as a whole, this work illustrates that small, unpredicted changes in the amino acid sequence of even large enzymes can have dramatic effects on their thermostability and/or activity.

Vitamin C (L-ascorbic acid) has a major biological role as a natural antioxidant. Aspirin belongs to the nonsteroidal anti-inflammatory drugs and functions as an antioxidant via its ability to scavenge-OH radicals. Bovine serum albumin (BSA) is the major soluble protein constituent of the circulatory system and has many physiological functions including transport of a variety of compounds. In this report, the competitive binding of vitamin C and aspirin to bovine serum albumin has been studied using constant protein concentration and various drug concentrations at pH 7.2. FTIR and UV–Vis spectroscopic methods were used to analyze vitamin C and aspirin binding modes, the binding constants and the effects of drug complexation on BSA stability and conformation. Spectroscopic evidence showed that vitamin C and aspirin bind BSA via hydrophilic interactions (polypeptide and amine polar groups) with overall binding constants of Kvitamin C-BSA = 1.57 × 104 M−1 and Kaspirin-BSA = 1.15 × 104 M−1; assuming that there is one drug molecule per protein. The BSA secondary structure was altered with major decrease of α-helix from 64% (free protein) to 57% (BSA-vitamin C) and 54% (BSA-aspirin) and β-sheet from 15% (free protein) to 6–7% upon drug complexation, inducing a partial protein destabilization.► The binding constant of vitamin C-BSA was measured higher than aspirin-BSA. ► At low drugs concentration, no major protein conformational changes occur. ► At high drugs contents, significant decrease of secondary structures was observed. ► A partial protein destabilization was occurred at high drug concentrations. ► BSA can be considered as a good carrier for vitamin C and aspirin in vitro.

A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins, and protecting them from various kinds of stress. Most of them are sugars, ...

Aims: The aim of this study was to investigate whether the shelf-life of diagnostic antibodies is longer than the expiry date on the label. Methods and results: Four independent laboratories tested a small number of diagnostic antibodies kept at +4°C for 12-26 years, and found them to work perfectly on routine histology sections. Conclusions: Diagnostic antibodies may have a workable half-life in excess of 10 years, and the emphasis on performance should shift to the preservation of antigenic targets in the tissue.

The fl uorescence thermal shift (FTS) method is a biophysical technique that can improve productivity in a structural genomics pipeline and provide a fast and easy platform for identifying ligands in protein function or drug discovery screening. The technique has gained widespread popularity in recent years due to its broad-scale applicability, throughput, and functional relevance. FTS is based on the principle that a protein unfolds at a critical temperature that depends upon its intrinsic stability. A probe that will fl uoresce when bound to hydrophobic surfaces is used to monitor protein unfolding as temperature is increased. In this manner, conditions or small molecules that affect the thermal stability of a protein can be identifi ed. Herein, principles, protocols, data analysis, and special considerations of FTS screening as performed for the Center for Structural Genomics of Infectious Diseases (CSGID) pipeline are described in detail. The CSGID FTS screen is designed as a high-throughput 384-well assay to be performed on a robotic platform; however, all protocols can be adapted to a 96-well format that can be assembled manually. Data analysis can be performed using a simple curve fi tting of the fl uorescent signal using a Boltzmann or double Boltzmann equation. A case study of 100 proteins screened against Emerald Biosystem's ADDit™ library is included as discussion.

The effect of homogenization and surface-active stabilizers (0-1 wt% sodium caseinate, whey protein isolate, sodium dodecyl sulfate, or polyoxyethylene sorbitan monolaurate) on the microstructure and colloidal stability of coconut milk was determined using measurements of particle size and creaming, as well as microscopy. The freshly prepared coconut milk emulsions (1.8-2% protein, 17-18% fat) had large (d 43 $10 mm) but non-flocculated droplets. Homogenization reduced the primary droplet size but induced flocculation. Adding small-molecule surfactants after the homogenization step can displace coconut proteins from the interface and break up these flocs, but adding them before homogenization increased the efficiency of the homogenization step and produced stable, submicron-sized emulsion droplets. Adding protein stabilizers did not break up the flocs of coconut milk droplets when added after homogenization, but did increase the efficacy of the homogenization step when added prior to it. Adding stabilizers to non-homogenized coconut milk had no effect on the structure or properties of the emulsions. r

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.