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At least 19 recordsLinked to original sources

PubMed2006

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

Animals↗

PubMed1993

Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability.

We have chosen two members of the microbial RNase family, barnase and binase, which have 85% identity (17 substitutions and 1 deletion) and almost identical three-dimensional structure, to study the evolution of protein stability. The 17 residues that differ are scattered throughout the molecule. Each of the 17 differing residues has been mutated independently and the effect on protein stability analysed. Each point mutation has an effect on protein stability that ranges from +1.1 to -1.1 kcal mol-1. These changes in energy are additive. There is no clear correlation between the type of mutation and the effect on protein stability. A multiple mutant having six of the single mutations that increase the stability of barnase is 3.3 kcal mol-1 more stable than wild type and has the same activity. There could be selective pressure to maintain proteins at a certain stability and, consequently, mutations that decrease stability tend to be counterbalanced by stabilizing mutations. Alternatively, there could simply be pressure to maintain stability above a certain level, and any further increases in stability need not be maintained during evolution. These results suggest a simple way to improve the stability of proteins: choose two homologous proteins that have high similarity, mutate individually all of the residues that differ between the two, and combine the mutations that increase the stability in a multiple mutant.

Amino Acid Sequence↗

PubMed1998

What ultrastable globular proteins teach us about protein stabilization.

Proteins, due to their delicate balance of stabilizing and destabilizing interactions, are only marginally stable if physiological conditions are considered as the standard state. Enhanced intrinsic stability of "ultrastable" proteins, e.g., from extremophiles, requires only minute local structural changes. Thus, general strategies of stabilization are not available for temperature, pH, salt, or pressure adaptation. Mechanisms of enhanced thermal stability involve improved packing or docking of structural elements (domains, subunits), as well as specific local interactions, e.g., networks of ion pairs. Relating the structure and stability of eye lens crystallins (which do not undergo any turnover during the life time of an organism), point mutations, nicking and swapping of domains, grafting of linker peptides between domains, and denaturation-renaturation allowed the cumulative nature of protein stability and its relation to the hierarchy of protein structure and folding to be established. In this review, recent results for crystallins and enzymes from hyperthermophiles will be discussed as models to illustrate mechanisms of protein stabilization.

Hydrogen-Ion Concentration↗

PubMed1998

Coupling protein stability and protein function in Escherichia coli CspA.

BACKGROUND: CspA is a small protein that binds single-stranded RNA and DNA. The binding site of CspA consists of a cluster of aromatic amino acids, which form an unusually large nonpolar patch on the surface of the protein. Because nonpolar residues are generally found in the interiors of proteins, this cluster may have evolved to bind nucleic acids at the expense of protein stability. RESULTS: Three neighboring phenylalanines have been mutated singly and in combination to leucine and to serine. All mutations adversely affect DNA binding. Surprisingly, all mutations, and especially those to serine, are destabilizing. CONCLUSIONS: The aromatic cluster in CspA is required not only for protein function but also for protein stability. This result is pertinent to the design of beta-sheet proteins and single-stranded nucleic acid binding proteins, whose binding mode is proposed to be of aromatic-aromatic intercalation.

Bacterial Proteins↗

PubMed1994

Physical stability and protein stability of freeze-dried cakes during storage at elevated temperatures.

The relationship between physical stability of freeze-dried cakes and protein stability during storage was studied using beta-galactosidase as a model protein and inositol as an excipient. Amorphous samples freeze-dried from solutions containing the enzyme and various concentrations of inositol in sodium phosphate buffer (50 mM, pH 7.4) were stored for 7 days over P2O5 at 40 to 70 degrees C. Structural collapse and inositol crystallization were observed in some of the samples, depending on the formulation and storage temperature. The physical stability of freeze-dried samples was also studied by differential scanning calorimeter (DSC). Inositol showed a protein-stabilizing effect when its amorphous form was retained during storage, regardless of structural collapse. However, crystallization of inositol during storage removed its stabilizing effect. Addition of water-soluble polymers such as dextran, Ficoll and carboxymethyl cellulose sodium salt (CMC-Na) preserved activity of the enzyme by preventing inositol crystallization.

Aspergillus oryzae↗

PubMed2001

Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability.

Protein thermal dynamics was evaluated by neutron scattering for halophilic malate dehydrogenase from Haloarcula marismortui (HmMalDH) and BSA under different solvent conditions. As a measure of thermal stability in each case, loss of secondary structure temperatures were determined by CD. HmMalDH requires molar salt and has different stability behavior in H(2)O, D(2)O, and in NaCl and KCl solvents. BSA remains soluble in molar NaCl. The neutron experiments provided values of mean-squared atomic fluctuations at the 0.1 ns time scale. Effective force constants, characterizing the mean resilience of the protein structure, were calculated from the variation of the mean-squared fluctuation with temperature. For HmMalDH, resilience increased progressively with increasing stability, from molar NaCl in H(2)O, via molar KCl in D(2)O, to molar NaCl in D(2)O. Surprisingly, however, the opposite was observed for BSA; its resilience is higher in H(2)O where it is less stable than in D(2)O. These results confirmed the complexity of dynamics-stability relationships in different proteins. Softer dynamics for BSA in D(2)O showed that the higher thermostability is associated with entropic fluctuations. In the halophilic protein, higher stability is associated with increased resilience showing the dominance of enthalpic terms arising from bonded interactions. From previous data, it is suggested that these are associated with hydrated ion binding stabilizing the protein in the high-salt solvent.

Animals↗

PubMed1991

Protein stability and protein folding.

Proteins show only marginal free energies of stabilization. Mutative adaptations to extremes of physical conditions (high temperature, pressure and salt concentration) tend to maintain 'corresponding states' regarding overall topology, flexibility and hydration. Since enhanced stability requires only minute local changes in the structure of a given protein, general strategies of adaptation cannot be established. Apart from alterations at the protein level, extrinsic factors such as ions, cofactors or specific ligands may serve to enhance in vivo and in vitro protein stability. Protein folding and association reflect the hierarchy of protein structure, with the formation of secondary/supersecondary structure, subdomains/domains and structured monomers as consecutive steps. The process requires highly specified environmental conditions; e.g. active mesophilic or halophilic proteins cannot be expressed in thermophilic and non-halophilic hosts. On the other hand, a given protein may tolerate extreme sequence variability without substantially altering its three-dimensional structure and stability. Significant rate-determining steps in the overall reaction, that is, formation of disulphide bridges, proline isomerization and oligomerization, are catalysed by specific enzymes or directed by 'helper proteins' (protein disulphide isomerase, peptidyl-prolyl cis-trans isomerase and chaperones). Physiological stress conditions, (site-directed) mutations, and in vitro studies may be used to unravel the significance of the three 'shuffling reactions'.

Amino Acid Isomerases↗

PubMed1999

The effect of Glu75 of staphylococcal nuclease on enzyme activity, protein stability and protein unfolding.

Staphylococcal nuclease mutants, E57G and E75G, were generated. A comparison of the kinetic parameters both for mutants and wild-type protein shows that the Michaelis constants (Km) were almost identical for the wild-type protein and E57G mutant. An approximately 30-fold decrease in Km compared with the wild-type protein was observed for the E75G mutant. The turnover numbers for the enzyme (kcat) were higher with both the wild-type protein and the E57G mutant (3.88 +/- 0.21 x 103 s-1 and 3.71 +/- 0.28 x 103 s-1) than with the E75G mutant (3.04 +/- 0.02 x 102 s-1). The results of thermal denaturation with differential scanning microcalorimetry indicate that the excess calorimetric enthalpy of denaturations, DeltaHcal, was almost identical for the wild-type protein and E57G mutant (84.1 +/- 6.2 kcal.mol-1 and 79.3 +/- 7.1 kcal.mol-1, respectively). An approximately twofold decrease in DeltaHcal compared with the wild-type protein was observed for the E75G mutant (42.7 +/- 5.5 kcal.mol-1). These outcomes imply that Glu at position 75 plays a significant role in maintaining enzyme activity and protein stability. Further study of the unfolding of the wild-type protein and E75G mutant was conducted by using time-resolved fluorescence with a picosecond laser pulse. Two fluorescent lifetimes were found in the subnanosecond time range. The faster lifetime (tau2) did not generally vary with either pH or the concentration of guanidinium hydrochloride (GdmHCl) in the wild-type protein and the E75G mutant. The slow lifetime (tau1), however, did vary with these parameters and was faster as the protein is unfolded by either pH or GdmHCl denaturation. The midpoints of the transition for tau1 are pH 3.5 and 5.8 for the wild-type protein and E75G mutant, respectively, and the GdmHCl concentrations are 1.1 m and 0.6 m for the wild-type protein and E75G mutant, respectively. Parallel steady-state fluorescence measurements have also been carried out and the results are in general agreement with the time-resolved fluorescence experiments, indicating that Glu at position 75 plays an important role in protein unfolding.

Calorimetry, Differential Scanning↗

PubMed2003

Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose.

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.

Animals↗

PubMed1993

Structural thermodynamics: prediction of protein stability and protein binding affinities.

The relationships between the structure of a protein and the thermodynamic parameters that determine its stability constitute the focus of attention of an increasing number of research laboratories. Advances in this area have reached the point in which accurate protein stability predictions and interaction energetics based on crystallographic information appear to be within reach. The purpose of this article is to summarize these developments and discuss future goals and trends.

Drug Stability↗

PubMed1990

Strength and co-operativity of contributions of surface salt bridges to protein stability.

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.

Alanine↗

PubMed2002

The effect of protein stability on protein-monoglyceride interactions.

We have previously shown that proteins such as beta-lactoglobulin and lysozyme insert into monoglyceride monolayers and are able to induce an L(beta) to coagel phase transition in monoglyceride bilayers. These studies gave a first indication that protein stability could be an important factor for these interactions. This study therefore aims at further investigating the potential role of protein stability on protein-monoglyceride interactions. To this end we studied the interaction of stable and destabilized alpha-lactalbumin with monostearoylglycerol. Our results show that protein stability is important for the insertion of proteins into a monostearoylglycerol monolayer, such that the lower the stability of the protein the better the protein inserts. In marked contrast to beta-lactoglobulin and lysozyme we found that destabilized alpha-lactalbumin does not induce the L(beta) to coagel phase transition in monoglyceride bilayers. We propose that this is due to an increased surface coverage by the protein which could result from the unfolding of the protein upon binding to the interface.

Calorimetry, Differential Scanning↗

PubMed2004

The DinI protein stabilizes RecA protein filaments.

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.

Adenosine Triphosphate↗

PubMed1995

A relationship between protein stability and protein function.

Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal.mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal.mol-1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.

Bacteriophage T4↗

PubMed2005

Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying.

Supercritical fluid (SCF) drying has been proposed as an alternative for freeze-drying to stabilize proteins. Here we studied the influence of sucrose and trehalose during SCF drying on the protein stability and the physical powder characteristics of lysozyme and myoglobin formulations. The results obtained with SCF drying were compared with the results after freeze-drying of the same solutions. Aqueous protein solutions, with or without sugar, were sprayed into a SCF mixture of carbon dioxide and ethanol. The dried products were analyzed by residual water measurements, scanning electron microscopy, X-ray powder diffraction and differential scanning calorimetry. After reconstitution the protein structure was studied by UV/VIS, circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assay (lysozyme). The SCF dried and freeze-dried formulations showed comparable water contents, but their physical properties were substantially different. All freeze-dried cakes were amorphous with fully preserved protein structure. SCF dried sucrose-containing formulations showed agglomerated crystalline particles, whereas SCF dried trehalose-containing formulations appeared to consist of amorphous spherical particles. Particle morphology of excipients-free proteins was protein specific. Nearly all SCF dried lysozyme could be readily reconstituted, but for myoglobin significant fractions of SCF protein did not dissolve, especially in the absence of sugars. Covalent aggregation was not observed for the two proteins. For the recovered soluble fractions, the secondary protein structure was preserved. The tertiary structure was preserved for lysozyme, but not entirely for myoglobin. Surprisingly, during SCF drying trehalose was less protective than sucrose for myoglobin.

Animals↗

PubMed2004

Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry.

Many methods exist for measuring and studying protein denaturation in vitro. However, measuring protein denaturation in cells under conditions relevant to heat shock presents problems due to cellular complexity and high levels of light scattering that interfere with optical techniques. A general method for measuring protein denaturation in cells using high sensitivity differential scanning calorimetry (DSC) is given. Profiles of specific heat (c(p) vs. temperature) are obtained providing information about transitions in cellular components including the denaturation of proteins. The specific approaches employed with erythrocytes, bacteria, and mammalian cells are described, and an identification of several features of the DSC profiles is given. Protein denaturation on the level of roughly 7-20% occurs for commonly used heat shocks in mammalian cells.

Animals↗

PubMed1996

Analysis of the effect of local interactions on protein stability.

BACKGROUND: Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability. RESULT: To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f). CONCLUSIONS: These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.

Amino Acid Sequence↗

PubMed2004

Protein stability in nanocages: a novel approach for influencing protein stability by molecular confinement.

Confinement of a protein in a small inert space and microviscosity are known to increase its thermodynamic stability in a way similar to the mechanisms that stabilize protein fold in the cell. Here, to examine the influence of confinement on protein stability we choose four test cases of single domain proteins characterized by a wide range of melting temperatures, from approximately 73 degrees C of titin I27 to approximately 36 degrees C of yeast frataxin. All proteins are stabilized when confined in the gel, the most dramatic stabilization being that of yeast frataxin, whose melting temperature increased by almost 5 degrees C in the gel. In addition to being simple to use, this approach allows us to change the viscosity of the solvent without changing its composition or altering the structure of the proteins. The dimensions of the pores of the gels fall in the nanometer range, hence they are similar to those of the chaperone cavity. This method could therefore be used as a novel and powerful approach for protein folding studies.

Acrylic Resins↗