Buried surface area, conformational entropy, and protein stability.
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Amino Acids↗
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A novel mouse Smad4 mutation reduces protein stability and wild-type protein levels.
Smad4 is a key signal transducer of the transforming growth factor-beta (TGF-beta) superfamily of growth factors that are critical regulators of embryonic patterning and adult tissue homeostasis. The biological activity of the TGF-beta signaling is tightly controlled at multiple levels, including the abundance of SMAD4 proteins. We previously recovered a novel allele of Smad4 in a gene-based screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mouse embryonic stem cells. The mutation resulted in an unstable truncated protein that is degraded through proteasomal pathways. In the heterozygous state, this allele acts in a dominant negative fashion to reduce the wild-type protein level as well as signaling output. Biochemical characterization indicated that the truncated protein is able to form a complex with the wild-type protein, thus targeting it for proteasomal degradation as well. Phenotypic analyses of the heterozygous animals provided insight into the threshold requirement of Smad4-dependent signaling in vivo.
Alleles↗
Nucleotide sequence of the Mn-stabilizing protein gene of the thermophilic cyanobacterium Synechococcus elongatus.
The nucleotide sequence of the psbO gene encoding the extrinsic 33 kDa protein (the Mn-stabilizing protein) from the thermophilic cyanobacterium Synechococcus elongatus was determined. The deduced amino acid sequence consisted of 272 residues; 26 for the signal peptide and 246 for the mature protein. The amino acid sequences of nine proteolytic peptides from the isolated protein completely agreed with the deduced amino acid sequence. Several unique variations of amino acids were found in the primary structure, of which some may be related to the high thermostability of the protein.
Amino Acid Sequence↗
The manganese stabilizing protein (MSP) and the control of O2 evolution in the unicellular, diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142.
The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 temporally separates N2 fixation from photosynthesis. To better understand the processes by which photosynthesis is regulated, we have analyzed Photosystem (PS) II O2 evolution and the PSII lumenal proteins, especially the Mn stabilizing protein (MSP). We describe a procedure using glycine betaine to isolate photosynthetic membranes from Cyanothece sp. that have high rates of PSI and PSII activity. Analysis with these membranes demonstrated similar patterns of O2 evolution in vivo and in vitro, with a trough at the time of maximal N2 fixation and with a peak in the late light period. The pattern of PSI activity was also similar in vivo and in vitro. We cloned the genes for MSP (psbO) and the 12 kDa protein (psbU) and analyzed their transcriptional properties throughout the diurnal cycle. We suggest that the changes in PSII activity in Cyanothece sp. were due to conformational changes in a highly flexible MSP, a suggestion which can now be studied in a chimera. The Cyanothece sp. psbO gene has been transformed into Synechocystis sp. PCC 6803; MSP and O2 evolution in the resulting transformant had properties that were similar to those in Cyanothece sp., providing additional confirmation for the properties of Cyanothece sp. MSP.
Amino Acid Sequence↗
Protein stability: the COP9 signalosome gets in on the act.
The COP9 signalosome is a multiprotein complex somewhat similar to the lid component of the 26S proteasome. Recent studies suggest that it regulates the stability of proteins by interfering with the ubiquitin-proteasome pathway via deneddylation and phosphorylation.
COP9 Signalosome Complex↗
Charge-charge interactions influence the denatured state ensemble and contribute to protein stability.
Several recent studies have shown that it is possible to increase protein stability by improving electrostatic interactions among charged groups on the surface of the folded protein. However, the stability increases are considerably smaller than predicted by a simple Coulomb's law calculation, and in some cases, a charge reversal on the surface leads to a decrease in stability when an increase was predicted. These results suggest that favorable charge-charge interactions are important in determining the denatured state ensemble, and that the free energy of the denatured state may be decreased more than that of the native state by reversing the charge of a side chain. We suggest that when the hydrophobic and hydrogen bonding interactions that stabilize the folded state are disrupted, the unfolded polypeptide chain rearranges to compact conformations with favorable long-range electrostatic interactions. These charge-charge interactions in the denatured state will reduce the net contribution of electrostatic interactions to protein stability and will help determine the denatured state ensemble. To support this idea, we show that the denatured state ensemble of ribonuclease Sa is considerably more compact at pH 7 where favorable charge-charge interactions are possible than at pH 3, where unfavorable electrostatic repulsion among the positive charges causes an expansion of the denatured state ensemble. Further support is provided by studies of the ionic strength dependence of the stability of charge-reversal mutants of ribonuclease Sa. These results may have important implications for the mechanism of protein folding.
Enzyme Stability↗
Probing protein stability with unnatural amino acids.
Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (i) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (ii) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (iii) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.
Amino Acids↗
Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution.
The photosystem II (PSII) reaction center complex coordinates a cluster of Mn atoms that are involved in the accumulation of oxidizing equivalents generated by light-induced charge separations within the intrinsic portion of the PSII complex. A 33-kDa extrinsic protein, termed the Mn-stabilizing protein (MSP), has been implicated in the stabilization of two of the four Mn atoms of the cluster, yet the precise role of this protein in O2 evolution remains to be elucidated. Here we describe the construction of a mutant of the cyanobacterium Synechocystis sp. PCC6803 in which the entire gene encoding MSP has been deleted. Northern and immunoblot analyses indicate that other PSII proteins are expressed and accumulated, despite the absence of MSP. Fluorescence emission spectra at 77 K indicate PSII assembles in the mutant, but that the binding of MSP is required for the normal fluorescence characteristics of the PSII complex, and suggest a specific interaction between MSP and CP47. Fluorescence induction measurements indicate a reduced rate of forward electron transport to the primary electron donor, P680, in the mutant. It is concluded that in contrast to previous reports, MSP is not required for the assembly of active PSII complexes nor is it essential for H2O-splitting activity in vivo.
Blotting, Northern↗
Polymer models of protein stability, folding, and interactions.
The unfolded state and flexible linkers in the folded structure play essential roles in protein stability and folding and protein-protein interactions. Intrinsic to these roles is the fact that unfolded proteins and flexible linkers sample many different conformations. Polymer models may capture this and complement experiments in elucidating the contributions of the unfolded state and flexible linkers. Here I review what can be predicted from these models and how well these predictions match experiments. For example, Gaussian chain models give quantitatively reasonable predictions of the effects of residual charge-charge interactions in the unfolded state and qualitatively reasonable results for the effects of spatial confinement and macromolecular crowding on protein stability. A wormlike chain model has met with success in quantifying the effects of flexible linkers in binding affinity enhancement and in regulatory switches. In future developments, more realistic models may emerge from molecular dynamics simulations, and these models will guide experiments to advance our understanding of the unfolded state and flexible linkers.
Animals↗
Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix?
The purpose of this study is to investigate the mechanism of protein stabilization by sugars in the solid state. That is, explore whether the stabilization is controlled by "glass dynamics" or by native structure preservation through "specific interaction" between sugars and protein. The IgG1 antibody (150 kD) and recombinant human serum albumin (rHSA) (65 kD) were formulated with sorbitol, trehalose, and sucrose. Degradation of lyophilized formulations was quantified using size exclusion (SEC) and ion-exchange chromatography (IEX). The secondary structure of the protein in these formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy. The molecular mobility, as measured by the stretched relaxation time (tau(beta)) was obtained by fitting the modified stretched exponential (MSE) equation to the calorimetric data from the Thermal Activity Monitor (TAM). Compared with sucrose and trehalose, sorbitol could only slightly protect the protein against aggregation and had no effect on chemical degradation. The chemical degradation and aggregation rates of the protein decreased when the weight ratio of sucrose to protein increased from 0 to 2:1. Storage stability of the proteins showed a reasonably good correlation with the degree of retention of native structure of protein during drying as measured by the spectral correlation coefficient for FTIR spectra. The plots of tau(beta) as a function of fraction of sucrose passed through a maximum at 1:1 weight ratio of sucrose to protein. That is, the molecular mobility did not correlate with the stability of protein at high levels of sucrose content. Although the glass transition appears to be an important parameter for stability, protein stabilization by sugars in the solid state cannot be wholly explained by the glass dynamics mechanism, at least as measured by tau(beta). However, it is possible that the beta-relaxations rather than the alpha-relaxations (i.e., the tau we measured) are critical to stability. The data show that storage stability correlates best with "structure" as determined by FTIR spectroscopy. However, while a specific interaction between stabilizer and protein might be responsible for the preservation of native structure, the evidence supporting this position is not compelling.
Algorithms↗
Influence of specific regions in Lp82 calpain on protein stability, activity, and localization within lens.
PURPOSE: To determine the influence of specific regions within Lp82 calpain on protein stability, enzymatic activity, and localization within lens and to test the influence of an Lp82 knockout mouse on normal maturational proteolysis in lens. METHODS: DNA constructs for Lp82 and Lp82-related proteins were subcloned into the pcDNA 3.1 vector. The constructs contained a substitution of the novel sequence (NS) region from p94 for the AX1 N-terminal region of Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient expression of these Lp82-related proteins was performed in COS-7 mammalian cells. Immunoblotting and casein zymography were used to measure protein stability and enzymatic activity of the expressed proteins. Homologous recombination was used to knock out p94 gene expression and p94 splice variants such as Lp82 and Lp85 in the lenses of 10-day-old mice. Confocal microscopy revealed the immunohistochemical localization Lp82 and Lp85 within lens. RESULTS: Insertion of IS1 into Lp82 resulted in a lack of stable protein and loss of enzymatic activity. In contrast, substitution of the NS region for AX1 and insertion of IS2 into Lp82 had no effect on the stability of the Lp82-related proteins. p94 knockout mice at 10 days of age exhibited a total absence of Lp82 activity in the lens but normal activity for the separate mu- and m-calpain gene products. Calcium-induced in vitro proteolysis was retarded in these Lp82/p94 knockout lenses. Lp82 and Lp85 immunostaining was intense throughout the cytoplasm of the cortical and nuclear fibers of newborn mouse lenses with little staining in the epithelium. In contrast, immunostaining for the ubiquitous m-calpain was highest in the epithelium and bow region, with much lower levels in the nucleus. The naturally occurring IS3 insert in Lp85 also promoted the association of Lp85 with the perinuclear region of the nucleated lens fibers. CONCLUSIONS: The lack of the IS1 region in Lp82 accounts for the stability and abundance of enzymatically active Lp82 protein in rodent lenses. Conversely, the presence of the IS1 region is responsible for the lability of p94 and Rt88 calpains in muscle and retina, respectively. The insert in Lp85 may promote membrane association. A consequence of the specific loss of Lp82 in the lens may be to retard normal maturational proteolysis.
Animals↗
Identification of two serine residues important for p53 DNA binding and protein stability.
The p53 core DNA binding domain has been implied in Mdm2-mediated protein degradation. Here we show that the substitution of the serine residues 116 and 127 with alanine residues (S116/127A) has no effect on p53 DNA binding and protein stability. However, the substitution of the serine residues with the aspartic acid (S116/127D) abolished p53 DNA binding and led to protein stabilization. Importantly, we have shown that S116/127D exhibits a structural mutant conformation that results in a loss of p53-dependent transcription and Mdm2-mediated protein degradation.
Amino Acid Sequence↗
A novel method of determination of protein stability.
A novel method of determination of protein stability is described, which involves interfacial shear rheology of adsorbed protein layers. This technique provides information on the structural-mechanical properties of the adsorbed protein layers which can be related to: the rate of interfacial adsorption, interfacial interactions, and conformational changes in the adsorbed layers. The interfacial shear rheology of the blood proteins, bovine serum albumin and human immunoglobulin G was investigated. The air/aqueous and oil/aqueous interfaces were studied and the interfacial rheological activity of BSA was shown to be similar at three hydrophobic interfaces: air, squalene and mineral oil. The kinetics of interfacial film formation was shown to be time dependent, and aging effects were detected in both interfacial and bulk molecules. The absolute interfacial elasticity values decreased as the temperature increased. The protein solutions exhibited no interfacial rheological activity in the presence of the small surfactant molecules, Tween 80 and lecithin, under the conditions studied.
Chemical Phenomena↗
Exploiting elements of transcriptional machinery to enhance protein stability.
The correlation between protein structure and function is well established, yet the role stability/flexibility plays in protein function is being explored. Here, we describe an in vivo screen in which the thermal stability of a test protein is correlated directly to the transcriptional regulation of a reporter gene. The screen readout is independent of the function of the test protein, proteolytic resistance, solubility or propensity to aggregate indiscriminately, and is thus dependent solely on the overall stability of the test protein. The system entails the use of an engineered chimeric construct that consists of three covalently linked domains; a constant N-terminal DNA-binding domain, a variable central test protein, and a constant C-terminal transcriptional activation domain. The test proteins are mutant variants of the beta1 domain of streptococcal protein G that span fairly evenly a thermal stability range from as low as 38 degrees C to above 100 degrees C. When the chimeric construct contains a test variant of low thermal stability, the reporter gene is up-regulated to a greater extent relative to that of more stable/less flexible variants. A panel of nine Gbeta1 mutant variants was used to benchmark the screen, and spectroscopic methods were employed to characterize the thermal and structural properties of each variant accurately. The screen was combined with in silico methods to interrogate a library of randomized variants for selection of mutants of greater structural integrity.
Amino Acid Sequence↗
AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival.
p21(Cip1/WAF1) (p21), a p53-inducible protein, is a critical regulator of cell cycle and cell survival. p21 binds to and inhibits both the DNA synthesis regulator proliferating cell nuclear antigen and cyclin A/E-CDK2 complexes. Recently, p21 has also been shown to be a positive regulator of cell cycle progression as p21 is necessary for the assembly and activation of cyclin D1-CDK4/6 complexes. Furthermore, elevated p21 protein levels have been observed in various aggressive tumors as well as linked to chemoresistance. Here we demonstrate that p21 is directly phosphorylated by AKT/PKB, a survival kinase that is hyperactivated in many late stage tumors. Two sites (Thr(145) and Ser(146)) in the carboxyl terminus of p21 are phosphorylated by AKT/PKB in vitro and in vivo. Phosphorylation of Thr(145) inhibits PCNA binding, whereas phosphorylation of Ser(146) significantly increases p21 protein stability. Glioblastoma cell lines with activated AKT/PKB show enhanced p21 stability, and they are more resistant to taxol-mediated toxicity. Finally, AKT/PKB controls the assembly of cyclin D1-CDK4 complexes through modulation of p21 and cyclin D1 levels. These data imply that enhanced levels of p21 in tumors are due, in part, to phosphorylation by activated AKT/PKB. Furthermore, they suggest that one mechanism of AKT/PKB regulation of tumor cell survival and/or proliferation is to stabilize p21 protein.
Amino Acid Sequence↗
Cholesterol is critical for Epstein-Barr virus latent membrane protein 2A trafficking and protein stability.
Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking signals induced by the B cell receptor (BCR) altering normal B cell development. LMP2A specifically associates with Nedd4 family ubiquitin-protein ligases which downmodulate LMP2A activity by ubiquitinating LMP2A and LMP2A-associated protein tyrosine kinases (PTKs). Since specific ubiquitin tags provide an endocytic sorting signal for plasma membrane proteins which traffic to membrane vesicles, we examined LMP2A localization and trafficking. We found that LMP2A is secreted through exosomes, small endocytic membrane vesicles, as previously demonstrated for LMP1. Interestingly, the treatment of cells with methyl-beta-cyclodextrin (MCD), which depletes cholesterol from plasma membrane, dramatically increased LMP2A abundance and LMP2A exosome secretion. Cholesterol depletion also blocked LMP2A endocytosis resulting in the accumulation of LMP2A on plasma membrane. LMP2A phosphorylation and ubiquitination were blocked by cholesterol depletion. LMP2A in the exosomal fraction was ubiquitinated but not phosphorylated. These results indicate that cholesterol-dependent LMP2A trafficking determines the fate of LMP2A degradation.
Cell Line↗
Mapping tissue-specific genes correlated with age-dependent changes in protein stability and function.
Biophysical measurements indicative of protein stability and function were performed on crude extracts from liver, muscle, and lens of a genetically heterogeneous mouse population. Genetic information was used to search for quantitative trait loci (QTL) that influenced the biophysical traits, with emphasis on phenotypes that previously have been shown to be altered in aged animals. Spectroscopic and enzymatic assays of crude liver and muscle tissue extracts from approximately 600 18-month-old mice, the progeny of (BALB/cJxC57BL/6J)F1 females and (C3H/HeJxDBA/2J)F1 males, were used to measure the susceptibility of a ubiquitous glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to thermal denaturation. The rate constant for thermal inactivation of GAPDH correlated with markers on chromosome 5 (D5Mit79 and D5Mit251) for muscle lysates and chromosome 15 (D15Mit63 and D15Mit100) for liver tissue. The degree of variability of inactivation rate constants, a measure of the heterogeneity of muscle GAPDH in tissue extracts, was also associated with markers on chromosome 5 (D5Mit79 and D5Mit205). In addition, spectroscopic characteristics of extracted eye lens proteins were evaluated for their susceptibility to photooxidative stress. Absorbance and fluorescence emission characteristics of the lens proteins were mapped to QTL on chromosomes 5 and 15 (D5Mit25 and D15Mit171) while the degree of heterogeneity in photochemical oxidation kinetics was associated with a marker on the chromosome 8 (D8Mit42). Recent work has shown that GAPDH possesses a number of non-glycolytic functions including DNA/RNA binding and regulation of protein expression. Tissue specific differences in GAPDH stability may have significant consequences to these alternate functions during aging.
Aging↗
Improvement of interfacial protein stability by CHAPS.
Emulsification of aqueous protein solutions in methylene chloride triggered the formation of water-insoluble aggregates at a water/methylene chloride interface. As a result, the amounts of beta-lactoglobulin and ovalbumin recovered in water were 36 and 44%, respectively. Addition of 5 mM: CHAPS in the aqueous phase raised the degree of beta-lactoglobulin recovery to 96%. Sodium taurocholate, however, failed to improve protein recovery. The stabilizing effect of CHAPS was also protein-specific and concentration-dependent: at >or=5 mM: , the surfactant caused unfolding of ovalbumin to make a water-soluble oligomer. CHAPS thus stabilizes proteins at an interface.
Cholic Acids↗