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At least 487 records · Page 27Linked to original sources

PubMed2003

Solution structure of a circular-permuted variant of the potent HIV-inactivating protein cyanovirin-N: structural basis for protein stability and oligosaccharide interaction.

The high-resolution solution structure of a monomeric circular permuted (cp) variant of the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by NMR. Comparison with the wild-type (wt) structure revealed that the observed loss in stability of cpCV-N compared to the wt protein is due to less favorable packing of several residues at the pseudo twofold axis that are responsible for holding the two halves of the molecule together. In particular, the N and C-terminal amino acid residues exhibit conformational flexibility, resulting in fewer and less favorable contacts between them. The important hydrophobic and hydrogen-bonding network between residues W49, D89, H90, Y100 and E101 that was observed in wt CV-N is no longer present. For instance, Y100 and E101 are flexible and the tryptophan side-chain is in a different conformation compared to the wt protein. The stability loss amounts to approximately 2kcal/mol and the mobility of the protein is evident by fast amide proton exchange throughout the chain. Mutation of the single proline residue to glycine (P52G) did not substantially affect the stability of the protein, in contrast to the finding for wtCV-N. The binding of high-mannose type oligosaccharides to cpCV-N was also investigated. Similar to wtCV-N, two carbohydrate-binding sites were identified on the protein and the Man alpha1-->2Man linked moieties on the sugar were delineated as binding epitopes. Unlike in wtCV-N, the binding sites on cpCV-N are structurally similar and exhibit comparable binding affinities for the respective sugars. On the basis of the studies presented here and previous results on high-mannose binding to wtCV-N, we discuss a model for the interaction between gp120 and CV-N.

Amino Acid Sequence↗

PubMed2001

Free energies of protein decoys provide insight into determinants of protein stability.

We have calculated the stability of decoy structures of several proteins (from the CASP3 models and the Park and Levitt decoy set) relative to the native structures. The calculations were performed with the force field-consistent ES/IS method, in which an implicit solvent (IS) model is used to calculate the average solvation free energy for snapshots from explicit simulations (ESs). The conformational free energy is obtained by adding the internal energy of the solute from the ESs and an entropic term estimated from the covariance positional fluctuation matrix. The set of atomic Born radii and the cavity-surface free energy coefficient used in the implicit model has been optimized to be consistent with the all-atom force field used in the ESs (cedar/gromos with simple point charge (SPC) water model). The decoys are found to have a consistently higher free energy than that of the native structure; the gap between the native structure and the best decoy varies between 10 and 15 kcal/mole, on the order of the free energy difference that typically separates the native state of a protein from the unfolded state. The correlation between the free energy and the extent to which the decoy structures differ from the native (as root mean square deviation) is very weak; hence, the free energy is not an accurate measure for ranking the structurally most native-like structures from among a set of models. Analysis of the energy components shows that stability is attained as a result of three major driving forces: (1) minimum size of the protein-water surface interface; (2) minimum total electrostatic energy, which includes solvent polarization; and (3) minimum protein packing energy. The detailed fit required to optimize the last term may underlie difficulties encountered in recovering the native fold from an approximate decoy or model structure.

Biophysical Phenomena↗

PubMed1997

Packing as a structural basis of protein stability: understanding mutant properties from wildtype structure.

Modeling of protein core mutations using sidechain packing can forecast their effects on stability. We have assessed the structural basis of this approach, by evaluating the accuracy of our 1991 model of a three-site mutant of lambda repressor (V36L/M40L/V47I), against the recently reported crystal structure. The three mutated residues matched the crystal structure to within 0.89A (1.11A for sidechain atoms), giving fairly accurate sidechain placement and packing (81-99th percentile rank in coordinate accuracy). However, the model used different sidechain torsional angles than seen in the crystal structure at residues 36 and 40, apparently to compensate for the backbone shifts present in the actual mutant structure, but not accounted for in our modeling method. To understand the structural basis of stability across a set of lambda repressor core mutants, we have analyzed the mutant models, revealing several simple packing effects: V36I, predicted to be stabilized by filling a hydrophobic cavity; M40V, destabilized by a steric clash with the unusual structural demands of a helix-turn transition. These effects illustrate how mutant stability can often be understood directly from scrutiny of wildtype structure. Simply adding the calculated energies of neighboring point mutations predicts the stability effect of the combined mutant relatively well, with little apparent cooperativity, yielding simple rules for each site's amino acid preferences. Our treatment of core packing indicates that it can permit a large fraction of sequences to fit the native fold, as observed experimentally, far more than indicated by rotamer hard-sphere models.

Amino Acid Sequence↗

PubMed2005

The relationship between FRQ-protein stability and temperature compensation in the Neurospora circadian clock.

Temperature compensation is an important property of all biological clocks. In Neurospora crassa, negative-feedback regulation on the frequency (frq) gene's transcription by the FRQ protein plays a central role in the organism's circadian pacemaker. Earlier model calculations predicted that the stability of FRQ should determine the period length of Neurospora's circadian rhythm as well as the rhythm's temperature compensation. Here, we report experimental FRQ protein stabilities in frq mutants at 20 degrees C and 25 degrees C, and estimates of overall activation energies for mutant FRQ protein degradation. The results are consistent with earlier model predictions, i.e., temperature compensation of Neurospora's circadian rhythm is a highly regulated process where the stability of FRQ is an important factor in determining Neurospora's circadian period as well as the clock's temperature compensation. The partial loss of temperature compensation in frq7 and frq(S513I) mutants can be described by a simple negative-feedback model (the Goodwin oscillator) when the experimentally obtained activation energies of FRQ degradation for theses mutants are incorporated into the model.

Circadian Rhythm↗

PubMed1995

Biosynthetic incorporation of 7-azatryptophan into the phage lambda lysozyme: estimation of tryptophan accessibility, effect on enzymatic activity and protein stability.

The phage lambda lysozyme (lambda L) contains four tryptophans. These have been efficiently replaced by 7-azatryptophan (7aW) through biosynthetic incorporation into the overexpressed protein. Comparative analysis of the effect of temperature or pH on the fluorescence of the wild-type lambda L and 7aWs-containing protein (a lambda L) shows that the stability of the protein is only mildly reduced by 7aW incorporation above pH 5 but that it is strongly decreased below pH 4 on protonation of inaccessible 7aWs. The a lambda L fluorescence depends on pH as a consequence of its effect on the denaturation equilibrium, on the state of protonation of accessible 7aWs in the native state and of all 7aWs in the denatured state. The pH dependence of the fluorescence is used to estimate the number of accessible tryptophans in the protein. The result agrees with that derived from tryptophan NH exchange measurements by 1H-NMR. The acid limb of the activity-pH profile is characterized by a sharp drop that might arise from a cooperative acid-induced denaturation. The difference in acid stability of a lambda L versus lambda L is used to rule out this acid denaturation hypothesis as tryptophan replacement does not affect the lytic activity on chloroform-sensitized Escherichia coli cells or its pH profile.

Bacteriophage lambda↗

PubMed2004

A relationship between heme binding and protein stability in cytochrome b5.

Conformational changes and long-range effects are often observed in proteins when they associate with their ligands. In many cases, these structural perturbations are essential to function, and they are the result of complex networks of interactions. Here we used cytochrome b(5), a protein that undergoes extensive structural rearrangement upon heme binding, to seek a relationship between affinity for the cofactor and extent of refolding induced by its binding. Three variants of the water-soluble domain of the rat microsomal protein were chosen to affect the stability of the apoprotein or the holoprotein. Sequence alterations were introduced in the heme binding loop (type I mutations, D60R and (55)TENFED --> (55)TEPFEED, or PE), which is largely unstructured in the apoprotein state, and in the folded core of the apoprotein (type II mutation, P81A). Thermal and chemical denaturation experiments and heme transfer experiments were performed on these proteins. Type I mutations left the thermodynamic stability of the apoprotein unchanged. The first mutation (D60R) stabilized the holoprotein in a probable manifestation of enhanced helical propensity or improved electrostatic interactions. The second mutation (PE) decreased heme affinity and holoprotein stability in concert. For this protein, heme transfer experiments could be used to estimate the rate constant of heme loss from each of the heme orientational isomers. In contrast, the type II mutation resulted in a marked destabilization of the apoprotein but an intermediate effect on the holoprotein stability and heme affinity. These data supported that heme affinity could be modulated by the apoprotein stability and by specific residues remote from the heme binding site.

Animals↗

PubMed1988

The thermodynamics of protein stability. Cold destabilization as a general phenomenon.

A theoretical analysis of the temperature/stability profiles of proteins shows that, where a two-state model represents the denaturation, and where the free energy of denaturation delta G(T) shows a strong temperature dependence, then the protein becomes subject to both high- and low-temperature destabilization. In the simplest case delta G(T) is parabolic, therefore the high temperature TH, where delta (G(TH) = 0, is complemented by a low temperature TL, where delta G(TL) = 0. It is generally stated that the partial molal heat capacity change delta C accompanying the heat denaturation is positive and independent of the temperature. This implies that heating the protein through TL results in a negative delta C which seems physically unsatisfactory. The constant delta C model is explored and a physically more realistic model is advanced which allows for a temperature-dependent delta C which changes sign at some temperature within the range of stability of the native protein; delta G(T) then has the form of a skewed parabola. Experimental heat capacity data for native lysozyme and for a flexible polymer lend support to this model. The molecular basis of cold inactivation of proteins is discussed in the light of the thermodynamic analysis.

Cold Temperature↗

PubMed1983

Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding.

The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.

Amino Acid Sequence↗

PubMed1997

Degradative covalent reactions important to protein stability.

Commonly observed chemical modifications that occur in proteins during their in vitro purification, storage, and handling are discussed. Covalent modifications described include deamidation and isoaspartate formation, cleavage of peptide bonds at aspartic acid residues, cystine destruction and thiol-disulfide interchange, oxidation of cysteine and methionine residues, and the glycation and carbamylation of amino groups.

Aspartic Acid↗

PubMed2006

Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability.

Bet3 is a component of the transport protein particle complex involved in vesicular trafficking to and through the Golgi complex. X-ray structural analysis of human and mouse Bet3 revealed a hydrophobic tunnel inside the protein, which is occupied by a fatty acid linked to cysteine-68. We show here that Bet3 has strong self-palmitoylating activity. Incubation of purified Bet3 with [3H]palmitoyl-CoA (Pal-CoA) leads to a rapid and stoichiometric attachment of fatty acids to cysteine-68. Bet3 has an intrinsic affinity for Pal-CoA, and the palmitoylation reaction occurs at physiological pH values and Pal-CoA concentrations. Moreover, Bet3 is also efficiently palmitoylated at cysteine-68 inside vertebrate cells. Palmitoylation can occur late after Bet3 synthesis, but once the fatty acids are bound they are not removed, not even by disassembly of the Golgi complex. Narrowing the hydrophobic tunnel by exchange of alanine-82 with bulkier amino acids inhibits palmitoylation, both in vitro and inside cells, indicating that the fatty acid must insert into the tunnel for stable attachment. Finally, we show that palmitoylation of Bet3 plays a structural role. CD spectroscopy reveals that chemically deacylated Bet3 has a reduced melting temperature. As a consequence of its structural defect nonacylated Bet3 does not bind to TPC6, a further subunit of the transport protein particle complex, and is degraded inside cells.

Animals↗

PubMed2002

Modeling of denatured state for calculation of the electrostatic contribution to protein stability.

Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this article, a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution restricted by the protein sequence. The model was applied for the calculations of electrostatic interactions in two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, DeltaG(pH), reproduces the experimental data better than the commonly used null approximation (NA). It was shown that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pairwise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

Bacterial Proteins↗

PubMed1998

MEICPS: substitution mutations to engineer intracellular protein stability.

UNLABELLED: In MEICPS, results from earlier analyses are utilized to suggest possible substitution point mutations to engineer intracellular stability using a given sequence or structure of the protein. AVAILABILITY: From bvbreddy@ccmb.ap.nic.in. This program needs data from other software, PSA and SSTRUC, available from sali@tamika.rockefeller.edu and tom@cryst.bioc.cam.ac.uk, respectively. CONTACT: bvbreddy@ccmb.ap.nic.in

Computational Biology↗

PubMed1994

Reversed-phase liquid chromatography as a useful probe of hydrophobic interactions involved in protein folding and protein stability.

We have evaluated the potential of reversed-phase liquid chromatography (RPLC) as a probe of hydrophobic interactions involved in protein folding and stability. Our approach was to apply RPLC to a de novo designed model protein system, namely a two-stranded alpha-helical coiled coil. It was shown that the reversed-phase retention behaviour of various synthetic analogues of monomeric alpha-helices and dimeric coiled-coil structures correlated well with their stability in solution, as monitored by circular dichroism during guanidine hydrochloride and temperature denaturation studies. In addition, an explanation is offered as to why amphipathic coiled coils, an important structural motif in many biological systems, are more stable at low pH compared to physiological pH values. The results of this study suggest that not only may RPLC prove to be a useful and rapid complementary technique for understanding protein interactions, but also the de novo designed coiled-coil model described here is an excellent model system for such studies.

Amino Acid Sequence↗

PubMed1998

Site-specific mutations of the N-terminal threonines in the D1 protein affects photoautotrophic growth but not D1 protein stability in Synechocystis 6803.

The cyanobacterium Synechocystis 6803 was engineered to produce a D1 protein where one or more of the N-terminal threonines at positions 2, 3 and 4 were replaced by other amino acid residues. No phenotypic effects were found for the T2S or T2L mutations, whereas the T2V, T2L;T4V and T2V;T3V;T4V mutations resulted in reduced photoautotrophic growth rate and oxygen evolving activity. The mutant strain T2V;T3V;T4V exhibited an oxygen evolution activity that was only half of that for the wild-type strain. Despite of that, both accumulation and stability of the D1 protein in the thylakoid membrane appeared unaffected in the mutant.

Cyanobacteria↗

PubMed1991

Dipoles localized at helix termini of proteins stabilize charges.

The charge-stabilization effect associated with alpha-helices in proteins has been reexamined by microscopic calculations without any a priori assumptions about the dielectric constant of the protein. The calculations reproduce the observed charge stabilization effect of a helix in two well-defined test cases: a histidine residue situated at the C-terminal end of a helix in barnase and the sulfate ligand located near the N-terminal end of a helix of the sulfate-binding protein. They also show that the effective dielectric constant for helix-charge interactions is much larger than previously assumed and that the stabilizing effect of the helix is not associated with the helix macrodipole but rather with a few localized dipoles confined mostly to the first turn of the helix. It is predicted that mutations at one end of the helix should have very small effects on the stabilization of charges at the opposite terminus. It is pointed out that the relatively short-ranged effect of the helix is essentially similar to other cases in which localized dipoles play key roles in electrostatic stabilization.

Bacterial Proteins↗

PubMed2006

Dynamic unfolding of a regulatory subunit of cAMP-dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability.

Characterization of the unfolding dynamics of a recombinant type IA regulatory subunit (RIalpha) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RIalpha by CE in a buffer devoid of cAMP resulted in rapid dissociation of the complex from the original sample due to the high negative mobility of the ligand relative to receptor. This process enabled in-capillary generation of cAMP-stripped RIalpha, which was used to estimate the apparent dissociation constant (Kd) of 0.6 +/- 0.2 microM. A comparison of RIalpha dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RIalpha-cAMP) and without (i.e., cAMP-stripped RIalpha) excess cAMP in the buffer during electromigration. The presence of cAMP in the buffer confirmed greater stabilization of the protein, as reflected by a higher standard free energy change (DeltaG(U) degrees) of 10.1 +/- 0.5 kcal x mol(+1) and greater cooperativity in unfolding (m) of -2.30 +/- 0.11 kcal x mol(-1) M(-1). CE offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor-ligand complexes in free solution.

Cyclic AMP↗

PubMed2001

NMR relaxation studies of the role of conformational entropy in protein stability and ligand binding.

Recent advances in the measurement and analysis of protein NMR relaxation data have made it possible to characterize the dynamical properties of many backbone and side chain groups. With certain caveats, changes in flexibility that occur upon ligand binding, mutation, or changes in sample conditions can be interpreted in terms of contributions to conformational entropy. Backbone and side chain flexibility can either decrease or increase upon ligand binding. Decreases are often associated with "enthalpy-entropy compensation" and "induced fit" binding, whereas increases in conformational entropy can contribute to stabilization of complexes. In certain cases, conformational entropy appears to play a role in cooperative binding and enzyme catalysis. In addition, variations in conformational entropy and heat capacity may both be important in stabilizing the folded structures of proteins.

Entropy↗

PubMed1991

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability.

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗