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.     

Phenylalanine side chain behavior of the intestinal fatty acid-binding protein: the effect of urea on backbone and side chain stability

The equilibrium unfolding behavior of the intestinal fatty acid-binding protein has been investigated by (19)F-NMR after incorporation of 4-fluorophenylalanine and by pulsed field gradient diffusion (1)H-NMR. At low urea concentrations (0-3 m) but prior to the global unfolding that begins at 4 m urea, the protein exhibits dynamic motion in the backbone and an expanded hydrodynamic radius with no major change in the side chain orientation. As monitored by two-dimensional (19)F-(19)F nuclear Overhauser effect, the distance between two phenylalanine residues (Phe(68) and Phe(93)) located in the two different beta-sheets that enclose the internal cavity did not change up to 4 m urea. Additionally, the chemical shifts of these two residues changed almost identically as a function of denaturant. At all urea concentrations, as well as in the native protein, multiple conformations exist. These conformers interconvert at different rates under different conditions, ranging from slow exchange by showing separate peaks in the native state to intermediate exchange at intermediate urea concentrations. Residual structure persisted around Phe(62) even at very high concentrations of denaturant, suggesting that region as a nucleation site during folding. The results were compared with previous studies examining the backbone behavior (Hodsdon, M. E., and Frieden, C. (2001) Biochemistry 40, 732-742) and suggest that the side chains show more stability than the backbone prior to global unfolding of the protein.     

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.     

The fluorescent monomeric protein Kusabira Orange. Pressure effect on its structure and stability

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network.     

Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

Osmolyte effect on the stability and folding of a hyperthermophilic protein

Proteins are known to be stabilized by naturally occurring osmolytes such as amino acids, sugars, and methylamines. Here, we examine the effect of trimethylamine-N-oxide (TMAO) on the conformational stability of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII), which inherently possesses high conformational stability. Heat- and guanidine hydrochloride-induced unfolding experiments demonstrated that the conformational stability of Tk-RNase HII in the presence of 0.5M TMAO was higher than that in the absence of TMAO at all examined temperatures. TMAO affected the unfolding and refolding kinetics of Tk-RNase HII to a similar extent. These results indicate that proteins are universally stabilized by osmolytes, regardless of their robustness, and suggest a stabilization mechanism by osmolytes, caused by the unfavorable interaction of osmolytes with protein backbones in the denatured state. Our results also imply that the basic protein folding principle is not dependent on protein stability and evolution.     

The effect of protein complexation on the mechanical stability of Im9

Force mode microscopy can be used to examine the effect of mechanical manipulation on the noncovalent interactions that stabilize proteins and their complexes. Here we describe the effect of complexation by the high affinity protein ligand E9 on the mechanical resistance of the simple four-helical protein, Im9. When concatenated into a construct of alternating I27 domains, Im9 unfolded below the thermal noise limit of the instrument ( approximately 20 pN). Complexation of E9 had little effect on the mechanical resistance of Im9 (unfolding force approximately 30 pN) despite the high avidity of this complex (K(d) approximately 10 fM).     

Contribution of the hydrophobic effect to globular protein stability.

The decrease in conformational stability, delta(delta G), has been measured for 72 aliphatic side-chain mutants from four proteins in which a larger side-chain is replaced by a smaller side-chain so that steric effects are minimal. When these delta(delta G) values are corrected to the same accessibility, namely 100% buried, then the following -delta(delta G) values per -CH2- group (in kcal/mol) are obtained: Ile----Val (1.26), Ala (1.26), Gly (1.26); Leu----Ala (1.16), Gly (1.21); Val----Ala (1.23), Gly (1.53). The average of these values is 1.27(+/- 0.07) kcal/mol. The 72 individual values range from 0 to 2.4 kcal/mol with an average value of 1.27(+/- 0.51) (standard deviation) kcal/mol. When the delta Gtr values from n-octanol to water are corrected for the difference in volume between the solutes and the solvents, the average value for the same substitutions is 1.25(+/- 0.05) kcal/mol. This suggests that proteins gain 1.3(+/- 0.5) kcal/mol in stability for each -CH2- group buried in folding, and, furthermore, that the volume corrected delta Gtr values for n-octanol for the amino acid side-chains provide good estimates of the contribution of the hydrophobic effect to globular protein stability.     

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap

Bacteriocins are proteins secreted by many bacterial cells to kill related bacteria of the same niche. To avoid their own suicide through reuptake of secreted bacteriocins, these bacteria protect themselves by co-expression of immunity proteins in the compartment of colicin destination. In Escherichia coli the colicin M (Cma) is inactivated by the interaction with the Cma immunity protein (Cmi). We have crystallized and solved the structure of Cmi at a resolution of 1.95? by the recently developed ab initio phasing program ARCIMBOLDO. The monomeric structure of the mature 10kDa protein comprises a long N-terminal α-helix and a four-stranded C-terminal β-sheet. Dimerization of this fold is mediated by an extended interface of hydrogen bond interactions between the α-helix and the four-stranded β-sheet of the symmetry related molecule. Two intermolecular disulfide bridges covalently connect this dimer to further lock this complex. The Cmi protein resembles an example of a 3D domain swapping being stalled through physical linkage. The dimer is a highly charged complex with a significant surplus of negative charges presumably responsible for interactions with Cma. Dimerization of Cmi was also demonstrated to occur in vivo. Although the Cmi-Cma complex is unique among bacteria, the general fold of Cmi is representative for a class of YebF-like proteins which are known to be secreted into the external medium by some Gram-negative bacteria.     

The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.     

The effect on chromosome stability of deleting replication origins.

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.     

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.     

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability.

An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.     

The dark side of green fluorescent protein

Here, severe interference of chlorophyll with green fluorescent protein (GFP) fluorescence is described for medicago (Medicago truncatula), rice (Oryza sativa) and arabidopsis (Arabidopsis thaliana). This interference disrupts the proportional relationship between GFP content and fluorescence that is intrinsic to its use as a quantitative reporter. The involvement of chlorophyll in the loss of GFP fluorescence with leaf age was shown in vivo, by the removal of chlorophyll through etiolation or by ethanol extraction, and in vitro, by titration of a GFP solution with chlorophyll solutions of various concentrations. A substantial decrease in fluorescence in early development of medicago and rice leaves correlated with chlorophyll accumulation. In all three species tested, removal of chlorophyll yielded up to a 10-fold increase in fluorescence. Loss of GFP fluorescence in vitro was 4-fold greater for chlorophyll b than for chlorophyll a. Differences exist between plant species for the discrepancy between apparent GFP fluorescence and its actual level in green tissues. Substantial errors in estimating promoter activity from GFP fluorescence can occur if pigment interference is not considered.     

The effect of charge-introduction mutations on E. coli thioredoxin stability

Technological applications of proteins are often hampered by their low-stability and, consequently, the development of procedures for protein stabilization is of considerable biotechnological interest. Here, we use simple electrostatics to determine positions in E. coli thioredoxin at which mutations that introduce new charged residues are expected to lead to stability enhancement. We also obtain the corresponding mutants and characterize their stability using differential scanning calorimetry. The results are interpreted in terms of the accessibility in the native structure of the mutated residues and the potential effect of the mutations on the residual structure of the denatured state.