Probing the roles of conserved arginine-44 of Escherichia coli dihydrofolate reductase in its function and stability by systematic sequence perturbation analysis

Sequence perturbation analysis is a powerful method to reveal roles of an amino acid residue in function and stability of a protein. By using and improving this method, we studied roles of highly conserved Arg44 of Escherichia coli dihydrofolate reductase (DHFR) in its function and stability. Here, we introduced systematic amino acid substitutions at this position and found that all 19 kinds of amino acid substitutions were tolerated, but the mutations significantly reduced the enzymatic activity and the binding affinity toward the cofactor NADPH. Moreover, the mutational effects on the cofactor binding affinity were well correlated with those on the catalytic activity, indicating that the R44X mutations affect the catalytic activity mainly by modulating the cofactor binding affinity. On the other hand, thermal denaturation measurements showed that most mutations stabilized the protein. Comparison between the mutational effects and various amino acid indices taken from the AAindex database indicated that hydrophobicity and polarity are key determinants of amino acids favorable at this position. These results suggest that through electrostatic interactions Arg44 plays a functional role in retaining the cofactor binding affinity at the cost of the protein stability.     

Molecular stability of chicken and rabbit immunoglobulin G.

Molecular stability of chicken egg yolk immunoglobulin G (IgY) and that of rabbit IgG were compared by measuring antibody activities and conformational changes. Stability of rabbit IgG to acid denaturation was much higher than that of IgY. Conformation of the IgY molecule was readily changed in acidic conditions, resulting in a rapid loss of antibody activity. Much less stable natures of IgY to heat-treatment and guanidine-HCl denaturation than rabbit IgG were also observed. Differences in the structure between the two immunoglobulins that might participate in their different stability were inferred from their amino acid sequence data. Importance of the intramolecular disulfide linkage in the rabbit light chain and some other structural differences were suggested.     

Stabilization of hyperactive dihydrofolate reductase by cyanocysteine-mediated backbone cyclization

Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine- and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteine-mediated cyclization method is complementary to the intein-mediated cyclization method in stabilizing a protein without affecting its activity.     

Denaturation studies reveal significant differences between GFP and blue fluorescent protein

Green fluorescent protein (GFP) is an unusually stable fluorescent protein that belongs to a family of related auto-fluorescent proteins (AFPs). These AFPs have been generated from jellyfish GFP by mutating the amino acids in the chromophore or its vicinity. Variants that emit light in the blue region (Blue Fluorescent Protein, BFP), red region, or yellow region are readily available and are widely used in diverse applications. Previously, we had used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with SDS, urea, and heat. Surprisingly, we found that SDS, urea or heat, did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5, however, at pH 6.5, the protein lost all fluorescence within a very short period of time. These results suggested that GFP undergoes a structural/stability shift between pH 6.5 and 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat. In the present study, we wanted to explore whether the stability or structure of the closely related BFP is also pH dependent. As expected, we found heat-induced denaturation and renaturation of BFP to be pH dependent, very much like GFP. However, when exposed to other denaturants like urea/heat or SDS we found BFP to behave very differently than GFP. Unlike GFP, which at pH 8.5 and 7.5 is very resistant to SDS-induced denaturation, BFP readily lost about 20% of its fluorescence at pH 8.5 and about 60% fluorescence at pH 7.5. Also, our denaturation and renaturation studies show that under certain conditions, BFP is more stable than GFP, such that under conditions where GFP is completely denatured, BFP still retained significant fluorescence. Taken together, our preliminary results show that despite being very similar in both amino acid sequences and overall structures, there may be subtle and important structural/conformational differences between BFP and GFP.     

A Monte Carlo Study of the Relative Stability of Protein Helical Forms

We present simulation results on a simple model to describe the hydrogen bonding in proteins with helical structures. The approximation distinguishes between ! helices, where each amino acid interacts with another one located four residues apart, 3 10 structures, where the number of amino acids in between is three, and the ? arrangement, in which that number is five. We found that the main features of the system are determined by the most stable structure (the ! helix) and that the other type of hydrogen bonds appears just below the denaturation temperature of the peptide. The probability of finding a 3 10 -type bond is greater at the beginning or at the end of the peptide chain, irrespectively of its length, while in short peptides the existence of those bonds increases appreciably the denaturation temperature, promoting stability. On the other hand, the temperature of denaturation decreases with the length of the peptide to reach a value independent of the number of amino acid residues.     

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.     

Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability

It is widely accepted that solvent-exposed sites in proteins play only a negligible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T(den) difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2 degrees C at pH 2.0. The DeltaH(den) versus T(den) plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (DeltaC(p,den) = 0.41 kcal* mole(-1) * K(-1); 1 cal = 4.18 J) leading to a DeltaG(den) difference of 2.1 kcal*mole(-1). Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (DeltaT(den) =7.1 degrees C). Selective reduction of the Cys14-Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences. A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein-stability-derived secondary structure propensity scales should be taken with more caution.     

Characteristics of a de novo designed protein.

A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryptophan fluorescence measurements. The GuHCl denaturation is well described by a 2-state model. The NMR spectra, the thermal denaturation curves, and the 1-anilino-8-naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature. The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy.     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.     

Prediction of protein mutant stability using classification and regression tool

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.     

A new scale for side-chain contribution to protein stability based on the empirical stability analysis of mutant proteins

The hydrophobicity scales for amino acid side chains based on the transfer Gibbs energy (DeltaG(trans)) of amino acids from non-aqueous phases to water have been widely used to estimate the contribution of buried side chains to the conformational stability of proteins. In this paper, we propose a new scale for the side-chain contribution to protein stability, which is derived from data on protein denaturation experiments using systematic and comprehensive mutant proteins. In the experiments, the contribution of some physical properties were quantitatively determined as parameters in a unique equation representing the stability change (DeltaDeltaG) of mutant proteins as a function of the structural changes due to the mutations. These parameters are able conveniently to provide a scale for the side-chain contribution to protein stability. This new scale also has the advantage over the previously reported hydrophobicity scales of residues with the contributions of hydrogen bonds or secondary structural propensity. It may find practical application in algorithms for the prediction of protein structures.     

N-terminal extension changes the folding mechanism of the FK506-binding protein

Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.     

Structural variation manipulates the differential oxidative susceptibility and conformational stability of apolipoprotein E isoforms

A growing amount of evidence implicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease (AD). It is now generally believed that the epsilon4 allele is associated with AD and the oxidative stress is more pronounced in AD. However, only limited data are available on apoE isoform-specificity and its relationship to both the oxidative susceptibility and conformational stability of apoE. In this article, we use site-directed mutagenesis to investigate the structural role of amino acid residue 112, which is the only differing residue between apoE3 and E4. We examine the structural variation manipulating the oxidative susceptibility and conformational stability of apolipoprotein E isoforms. Arg112 in apoE4 was changed to Ala and Glu. Previous research has reported that apoE4 is more susceptible to free radicals than apoE3. In protein oxidation experiments, apoE4-R112A becomes more resistant to free radicals to the same extent as apoE3. In contrast, apoE4-R112E becomes the most susceptible protein to free radicals among all the apoE proteins. We also examine the conformational stability and the quaternary structural change by fluorescence spectroscopy and analytical ultracentrifugation, respectively. ApoE3 and E4 show apparent three- and two-state unfolding patterns, respectively. ApoE4-R112A, similar to apoE3, demonstrates a biphasic denaturation with an intermediate that appears. The denaturation curve for apoE4-R112E, however, also displays a biphasic profile but with a slight shoulder at approximately 1.5M GdmCl, implying that an unstable intermediate existed in the denaturation equilibrium. The size distribution of apoE isoforms display similar patterns. ApoE4-R112E, however, has a greater tendency to dissociate from high-molecular-weight species to tetramers. These experimental data suggest that the amino acid residue 112 governs the differences in salt-bridges between these two isoforms and thus has a significant impact on the free radical susceptibility and structural variation of the apoE isoforms.     

Formation of circular polyribosomes in wheat germ cell-free protein synthesis system

We have compared the urea stability of the human aromatic amino acid hydroxylases (AAAHs), key enzymes involved in neurotransmitter biosynthesis and amino acid homeostasis. Tyrosine-, tryptophan- and phenylalanine hydroxylase (TH, TPH and PAH, respectively) were transiently activated at low urea concentrations and rapidly inactivated in >3 M urea. The denaturation of TH occurred through two cooperative transitions, with denaturation midpoints of 1.41+/-0.06 and 5.13+/-0.05 M urea, respectively. Partially denatured human TH (hTH) retained more of its secondary structure than human PAH (hPAH), and was found to exist as tetramers, whereas hPAH dissociated into dimers. Furthermore, the urea-induced aggregation of hPAH was 100-fold higher than for hTH. These results suggest that the denatured state properties of the AAAHs contribute significantly to the stability of these enzymes and their tolerance towards missense mutations.     

Temperature and solvent effects on reaction centers from Chloroflexus aurantiacus and Chromatium tepidum.

Temperature and solvent effects on reaction center structures were examined in two thermophilic photosynthetic bacteria, Chloroflexus aurantiacus and Chromatium tepidum, in order to gain insight into the interactions among the reaction center proteins and pigment systems. Thermal stability of the reaction centers was found to be proportional to the optimum growth temperature. Circular dichroism (CD) spectra in the 250-300 nm region indicated that thermal denaturation destroyed tertiary structures (helix-to-helix interactions or amino acid residue conformation) in the native reaction center, keeping helical structures intact. Absorption and circular dichroism spectral changes showed that alcohol denatured the so-called special pair and the accessory BChl a independently. The alcohol denaturation further indicates that the coordination between BChl a and amino acid residue in the protein is one of the important interactions maintaining the pigment organization of the reaction centers.     

Bound peptide-dependent thermal stability of major histocompatibility complex class II molecule I-Ek

We used differential scanning calorimetry to study the thermal denaturation of murine major histocompatibility complex class II, I-E(k), accommodating hemoglobin (Hb) peptide mutants possessing a single amino acid substitution of the chemically conserved amino acids buried in the I-Ek pocket (positions 71 and 73) and exposed to the solvent (position 72). All of the I-Ek-Hb(mut) molecules exhibited greater thermal stability at pH 5.5 than at pH 7.4, as for the I-Ek-Hb(wt) molecule, which can explain the peptide exchange function of MHC II. The thermal stability was strongly dependent on the bound peptide sequences; the I-Ek-Hb(mut) molecules were less stable than the I-Ek-Hb(wt) molecules, in good correlation with the relative affinity of each peptide for I-Ek. This supports the notion that the bound peptide is part of the completely folded MHC II molecule. The thermodynamic parameters for I-Ek-Hb(mut) folding can explain the thermodynamic origin of the stability difference, in correlation with the crystal structural analysis, and the limited contributions of the residues to the overall conformation of the I-Ek-peptide complex. We found a linear relationship between the denaturation temperature and the calorimetric enthalpy change. Thus, although the MHC II-peptide complex could have a diverse thermal stability spectrum, depending on the amino acid sequences of the bound peptides, the conformational perturbations are limited. The variations in the MHC II-peptide complex stability would function in antigen recognition by the T cell receptor by affecting the stability of the MHC II-peptide-T cell receptor ternary complex.     

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability.

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

Effect of calcium and phosphatidic acid binding on the C2 domain of PKC alpha as studied by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.     

Genetically engineered human interleukin-6 variant with enhanced stability

A recombinant human interleukin-6 mutant with enhanced conformational stability toward denaturant was obtained by site-specific mutagenesis. The clone was identified as having a single amino acid substitution of Lys70Glu. When urea-induced denaturation was monitored by the change in fluorescence intensity at 360 nm, Lys70Glu mutation shifted the midpoint of unfolding transition from 5.8 M (wild type) to 6.6 M urea. This mutation did not impair the biological activity.