1Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russia, 2Department of Biochemistry and Molecular Biology, Penn State University, College of Medicine, Hershey, PA 17033, 3Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80208, 4Center for Computational Biology and Bioinformatics, Department of Biochemistry and Molecular Biology, School of Medicine, Indiana University Purdue University, Indianapolis, IN 46202 and 6Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA
5 To whom correspondence should be addressed. E-mail: vuversky{at}iupui.edu
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Abstract |
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Keywords: -lactalbumin/calcium binding/electrostatic interactions/site-directed mutagenesis/thermal stability
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Introduction |
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Similar results were obtained with HAMLET (human -LA made lethal to tumor cells) or BAMLET (bovine
-LA made lethal to tumor cells), which is native
-LA converted in vitro to an apoptosis-inducing form of the protein when in a stoichiometric complex with oleic acid (Svensson et al., 1999
, 2000
). HAMLET was shown to trigger apoptosis in tumor and immature cells, but healthy cells were resistant. HAMLET passed through the cytoplasm to the nucleus and accumulated in the cell nucleus. In tumor cells in vivo, HAMLET co-localized with histones and perturbed the chromatin structure (Duringer et al., 2003
). HAMLET was found to bind histone HIII strongly and to lesser extent histones HIV and HIIB. The binding of histones by HAMLET impaired their interaction with DNA. Based on these observations, it was concluded that HAMLET interacts with histones and chromatin in tumor cell nuclei, locking the cells into the apoptotic pathway via an irreversible disruption of chromatin organization (Duringer et al., 2003
).
Recently, it was found that non-fatty acid-bound monomeric bovine and human -LAs interacted electrostatically with basic proteins, histones and positively charged polyamino acids as simple models of histone proteins (Permyakov et al., 2004
). Thus, complexation of
-LA with oleic acid is not required for the interaction with histone proteins. The intrinsic ability of
-LA to interact strongly with charged polymers suggests that the protein surface possesses some electrostatic properties which complement this interaction.
Mutations of surface charged residues in multi-subunit proteins can increase their stability. For example, mutation of Glu165 to Gln or Lys in tetrameric malate dehydrogenase caused a dramatic increase in thermal stability at pH 7.5 (increase by about 24°C) (Bjork et al., 2004). Remarkably, the crystal structures of the two mutants showed only minor structural changes localized in close proximity to the mutated residues, indicating that the observed stability changes were caused by subtle changes in the complex network of electrostatic interactions at the dimerdimer interface.
Minagawa et al. constructed a thermostable mutant of lactate oxidase (Minagawa et al., 2003). Their molecular modeling suggested that the substitution of Gly for Glu at position 160 reduced the electrostatic repulsion between the negative charges of Glu160 and Glu130 in the (ß/
)8 barrel structure, but thermal inactivation experiments on the five different single-mutant lactate oxidases at position 160 (E160A, E160Q, E160H, E160R and E160K) showed that it was the side-chain molecular volume of the residue at position 160 that was the primary contribution to the thermostability.
Protein engineering experiments on glycosyl hydrolase showed that the thermostabilization resulted as a consequence of numerous favorable ionic interactions in the 83124 sequence with the other parts of protein matrix that became more rigid and less accessible to thermally activated solvent molecules (Bismuto et al., 2003).
Seven hyper-stable multiple mutants of staphylococcal nuclease have been constructed by various combinations of eight different stabilizing single mutants (including mutations of negatively charged residuesD21N and D21K) (Chen et al., 2000). Their thermal denaturation midpoint temperatures were 12.622.9°C higher than that of the wild-type. The crystal structures of these mutants were solved at high resolution, yet no major structural changes were found, with most changes localized around the site of mutation. Rearrangements were observed in the packing of side chains in the major hydrophobic core, although none of the mutations were in the core. Surprisingly, detailed structural analysis showed that packing had improved, with the volume of the mutant hydrophobic core decreasing as protein stability increased. The authors believed that these results indicate that optimization of packing follows as a natural consequence of increased protein thermal stability and that good packing is not necessarily the proximate cause of high stability (Chen et al., 2000
). The mutants showed that increased numbers of electrostatic and hydrogen bonding interactions are not obligatory for large increases in protein stability. Based on the electrostatic energy calculations, it has been suggested that at least two of mutants, D21N and D21K, increase stability by removing unfavorable electrostatic interactions (Chen et al., 2000
).
The development of reliable methods for the prediction of result of mutations in protein requires the knowledge of the force field. This is a complex task that should take into account the delicate balance between the different energy terms that contribute to protein stability. The force fields usually use an effective physical energy function or they are based on statistical potentials where energies are derived from the frequency of residue or atom contacts in the protein database or they use empirical data obtained from experiments. For example, a computer algorithm, FOLDEF (for FOLD-X energy function), has been developed to provide a fast and quantitative estimation of the importance of the interactions contributing to the stability of proteins and protein complexes (Guerois et al., 2002). At the same time, the free energy of protein unfolding includes several contributions, some of which are difficult to estimate. For this reason, the predictive power of methods developed so far is still not as high as desired and researchers continue to suggest new approaches. Mozo-Villarias et al. have developed a thermostability criterion for a protein in terms of a quasi-electric dipole moment (contributed by its charged residues) defined for an electric charge distribution whose total charge is not zero (Mozo-Villarias et al., 2003
). It was found that minimization of the modulus of this dipole moment increased its thermal stability. In spite of these efforts to create methods for prediction of effects of mutations on stability of proteins, we still do not have reliable and simple approaches to solve this problem.
In the work presented here, the energies of chargecharge interactions in apo- and Ca2+-loaded -LA were calculated using a TanfordKirkwood algorithm with solvent accessibility correction or using the finite difference PoissonBoltzmann method. These continuum electrostatic models have been shown to capture successfully the interactions between ionizable residues on the protein surfaces (for reviews, see Klapper et al., 1986
; Gilson, 1995
; Elcock and McCammon, 1998
; Schaefer et al., 1998
; Schutz and Warshel, 2001
; Dong and Zhou, 2002
; Bashford, 2004
; Dominy et al., 2004
; Feig et al., 2004
; Garcia-Moreno and Fitch, 2004
). The analysis revealed that several residues in the
-LA sequence have unfavorable chargecharge interactions. The theoretical predictions were confirmed by site-directed mutagenesis of those charged surface residues and detailed physico-chemical characterization of each mutant protein. The experimental data obtained were in good qualitative agreement with the theoretical predictions.
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Materials and methods |
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Bovine -LA (lot 60K7002) was purchased from Sigma Chemical (St. Louis, MO) and used without further purification. Protein concentrations were determined spectrophotometrically using an extinction coefficient E1%, 280 nm = 20.1 (Kronman et al., 1964
). EDTA standard solutions (Fisher Scientific) were used for calcium titration. All other chemicals were of reagent grade or higher. All solutions were prepared with distilled, deionized water.
Recombinant proteins were prepared as described previously (Anderson et al., 1997). The mutant plasmids were prepared by the method of Kunkel (Kunkel, 1985
). All proteins were characterized by absorption, fluorescence and CD spectroscopy.
All of the recombinant -LAs retained the additional N-terminal methionine residue which was previously found to contribute to both decreased thermal stability and lower calcium affinity (Ishikawa et al., 1998
; Chaudhuri et al., 1999
; Veprintsev et al., 1999
). Selective removal of the N-terminal Met was achieved using Aeromonas proteolitica aminopeptidase (Wilkes et al., 1973
; Prescott and Wilkes, 1976
) as described by Veprintsev et al. (1999)
. Only the amino terminal Met is removed as the activity of this aminopeptidase is stopped by N-terminal amino acids with large negatively charged side chain (i.e. the first residue, Glu1, in the native bovine
-LA sequence). Briefly, the digestion reaction was performed at a substrate to enzyme ratio of about 100:1 (
-LA
2 mg/ml, 2 h, 37°C, 10 mM HEPES, pH 8.0). Excess EDTA (final concentration, 10 mM) was added to quench the reaction. Proteins were separated by gel filtration on a Sephadex G-100 column and fractions containing
-LA were collected, dialyzed against 10 mM ammonium bicarbonate and lyophilized.
Saturation of intact and mutant -LA by calcium ions was achieved via addition of 1 mM CaCl2. The use of higher concentrations of calcium could result in saturation of the weaker secondary calcium-binding site of
-LA (Aramini et al., 1992
). Removal of Ca2+ was achieved via addition of 1 mM EDTA. Higher concentrations of EDTA could promote EDTA binding to
-LA (Permyakov et al., 1987
).
Instrumentation and methods
Fluorescence measurements
Fluorescence measurements were performed on a Perkin-Elmer LS50B or a laboratory-made instrument with a precision titrator device and Peltier temperature-controlled cell holder described previously (Permyakov et al., 1977). The excitation wavelength was 280.4 nm. All spectra were corrected and fit to log-normal curves using non-linear regression analysis (Marquardt, 1963
) to obtain the emission maximum for each spectrum (Burstein and Emelyanenko, 1996
). In all fluorescence experiments, illumination time and UV irradiation power levels were minimized in order to avoid UV-induced structural rearrangements (Vanhooren et al., 2002
; Permyakov et al., 2003
).
Temperature scans were performed stepwise, allowing the sample to equilibrate at each temperature for at least 5 min. Temperature was monitored directly inside the cell. The fraction of conversion from the native to the thermally unfolded state was calculated as described previously (Permyakov and Burstein, 1984; Permyakov, 1993
).
The calcium binding affinity of each -LA mutant was measured by spectrofluorimetric back-titration of the calcium-loaded protein with a strong calcium chelator (e.g. EDTA) at fixed pH. Calculations of the calcium association constant from the experimental data were based on a competitive binding scheme between protein (P) and chelator (H) for calcium ions (Permyakov et al., 1985
; Permyakov, 1993
):
![]() | (1) |
Calcium binding and protein stability
The role of calcium on stability is linked to the difference in binding affinity for the folded protein and the unfolded protein (Permyakov et al., 1985):
![]() | (2) |
![]() | (3) |
Because calcium has a much higher affinity for the folded state (i.e. K1 >> K0), the folding equilibrium of the calcium-loaded state is shifted towards higher temperatures (i.e. G1 >>
G0).
In principle, in the presence of calcium, the total concentration of the folded protein is the sum of the two species, [F] and [FCa] and similarly for the unfolded state, [U] and [UCa]. Thus, the overall equilibrium constant can be described as
![]() | (4) |
Scanning calorimetry
Scanning calorimetric measurements were carried out on a VP-DSC differential scanning microcalorimeter (Microcal, Northhampton, MA) at a 0.5 K/min or 1 K/min heating rate in 10 mM HEPESKOH buffer, pH 7.7. A pressure of 30 psi was maintained in order to prevent degassing of the solutions during heating. Protein concentrations were 0.31 mg/ml. The heat sorption curves were baseline corrected. Protein specific heat capacity (Cp) was calculated as described by Privalov and Potekhin (1986). The partial molar volume was calculated according to Hakel et al. (1999)
. The temperature dependence of Cp was fitted to a simple two-state model, assuming that the difference between heat capacities of the denatured and native proteins (
Cp) was independent of temperature (all values were normalized by molecular weight):
![]() | (5) |
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Here Cp,D is the specific heat capacity of the denatured protein, linearly extrapolated to the transition region. The fitting parameters, Cp,
HVH (van't Hoff's enthalpy of protein denaturation) and T0 (mid-transition temperature) were estimated with the Origin 5.0 software provided with the MicroCal VP-DSC. The free energy change of thermal denaturation,
G, was calculated as follows:
![]() | (6) |
Circular dichroism Circular dichroism measurements were performed on either an AVIV 62DS, Applied Photophysics PiStar or Jasco J-500A spectropolarimeter. Typical instrument conditions were scan rate 5 nm/min and time constant 8 s. The pathlength was 0.19 mm for the far-UV and 10 mm for the near-UV region. All data were baseline corrected.
Electrostatics calculations
The energies of chargecharge interaction were calculated using a TK-SA procedure, implementation of which is described in detail elsewhere (Ibarra-Molero et al., 1999; Loladze et al., 1999
; Makhatadze et al., 2003
). Briefly, the energy of pairwise interactions between unit charges was calculated according to a TanfordKirkwood algorithm (Tanford and Kirkwood, 1957
) with the solvent accessibility correction as proposed by Matthew and Gurd (1986)
. The mean field approximation was used for calculating the effect of chargecharge interactions on the pKa of ionizable groups from their model compound values (Asp, 4.0; Glu, 4.5; Lys, 10.5; Arg, 12.0; His, 6.3; Tyr, 10.5; N-terminus, 7.7; C-terminus, 3.6). Calculations were performed on the PDB entries 1F6R (apo-form) and 1F6S (Ca2+-bound form) for bovine
-LA (Chrysina et al., 2000
). Missing atoms were reconstructed using the default option of the SwissPDB Viewer. Calculations of the chargecharge interactions for each of the six structural subunits in each PDB file were performed and average values were reported. For comparison of native
-LA with the various protein mutants, it was assumed that the chargecharge interactions in the unfolded states of these proteins were similar. The results of TK-SA calculations were also compared with the calculation done using the finite difference PoissonBoltzmann (FDPB) method as implemented in the UHBD software package (Antosiewicz et al., 1994
) as described (Fitch et al., 2002
; Makhatadze et al., 2004
). Both methods gave qualitatively identical results (see below). In addition, chargecharge interactions in the unfolded state were calculated using a Gaussian chain model as suggested by Zhou (2002a)
. Because the residues of interest are located on the protein surface and have high (>50%) solvent accessibility, the effects of solvation were not explicitly taken into account.
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Results and discussion |
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Calculations of the energies of the chargecharge interactions were carried out for three -LA forms: apo-protein (1F6R), holo-protein (1F6S) and an apo-form based on the 1F6S structure with Ca2+ ion excluded from the calculations. Comparison of the results from these calculations (Figure 1) reveals several interesting features:
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Based on these observations, one can predict that mutations, which neutralize side chains that are involved in unfavorable chargecharge interactions (e.g. Glu1, Glu7, Glu11), should result in an increase in protein stability. Conversely, substitutions leading to charge neutralization on side chains that provide favorable chargecharge interactions (i.e. Asp14, Glu25) should result in a decrease in protein stability relative to the native protein. Furthermore, from the analysis presented above, the most dramatic changes would be expected from mutations in the calcium-binding loop (including those non-coordinating residues) and secondly the N-terminal region of the protein.
The energies calculated for the calcium loop were not unexpected and the pronounced stabilization of the apo-form by Ca2+ binding originated from the negative charge compensation in the coordination site was demonstrated earlier (Permyakov et al., 2001). Until recently, however, the N-terminal region of
-LA had not been examined with respect to charged amino acid residues and protein stability. Consequently, we constructed a set of mutants covering this region in the 3-D structure of
-LA: E1Q, E7Q, E11L, D14N, D37N, including two additional mutants previously reported:
E1 (E1M) and E25A (Veprintsev et al., 1999
; Permyakov et al., 2000
). Since recombinant
-LA contains an extra methionine residue at the N-terminus, which was found to destabilize protein structure, thermal stability and calcium affinity (Ishikawa et al., 1998
; Chaudhuri et al., 1999
; Veprintsev et al., 1999
), selective removal of the additional N-terminal Met from recombinant proteins was performed using A.proteolitica aminopeptidase enzyme [with the exception of the mutants
E1 (E1M) and E1Q].
Physico-chemical properties of -lactalbumin mutants and comparisons with electrostatic calculations
The correctness of folding of -LA mutants is assessed, in part, from the maximum wavelength positions of the intrinsic fluorescence spectra (
max) of Ca2+-loaded (1 mM CaCl2)
-LA forms (see Table I). This spectral parameter reflects the mobility and polarity of the environment of emitting residues in proteins and generally reflects the degree of accessibility to solvent molecules (Permyakov, 1993
). All of the mutants studied here, except for desMet E25A, possess similar
max values, within 45 nm, which indicates only slight structural perturbations of the environment of their tryptophan residues. The increase of 89 nm in
max for desMetE25A
-LA reflects a significantly increased Trp exposure to solvent compared with native
-LA.
|
Typical spectrofluorimetric thermal denaturation curves for the apo- (1 mM EDTA, pH 7.7) and calcium-loaded (1 mM CaCl2) states of desMet-D37N -LA are shown in Figure 2. A red shift in the
max value with temperature corresponds to a progressive exposure of Trp residues to solvent water accompanying protein denaturation. It is remarkable that the D37N substitution shifts the thermal unfolding transition toward higher temperature, both in the absence and presence of calcium. Similar observations were found from scanning microcalorimetry (Figure 3).
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Table I summarizes all of the thermal transition mid-temperatures (tm) for the -LA species in this work. Every mutant studied possessed a thermal transition, even in the absence of calcium. Hence none of these mutations resulted in a total loss of protein tertiary structure. Yet desMet D14N
-LA displayed the most significant decrease in thermal stability, in both the absence and presence of calcium, resembling recombinant wt
-LA. A similar, but less pronounced, destabilization was observed for desMet E25A
-LA. On the other hand,
E1(E1M) and desMet D37N
-LA exhibited significantly increased thermal stabilities (see Table I). Overall, the charge-neutralizing mutations in the N-terminal region of
-LA resulted in protein forms differing in thermal stability by more than 17°C, suggesting very unusual electrostatic interactions in this region. Yet other substitutions that were predicted to increase thermal stability, namely desMet E7Q and desMet E11L, resulted in relatively minor effects (<2°C).
The results obtained with E1Q -LA are especially interesting. Although this mutant exhibited slightly lower thermal stability (13°C) with respect to native
-LA, this particular mutant contains an additional N-terminal Met residue, thus E1Q
-LA should be compared with recombinant wild-type
-LA. Consequently, removal of the negative charge at Glu1 in wild-type
-LA results in a substantial recovery of protein thermal stability. The same effect was observed for the native protein, i.e. substitution of Glu1 by Met1 in the
E1(E1M) mutant significantly increased the thermal stability of
-LA (Table I). The stabilizing effects of both mutations were easily rationalized from the electrostatic calculations in Figure 1, which showed that the carboxylate side chain of Glu1 provides distinctly unfavorable contributions; hence charge neutralization of the Glu side chain should add protein stabilization (as confirmed from the experimental data). Furthermore, electrostatic calculations for wild-type protein showed that the extra N-terminal Met residue resulted in a decrease in the favorable contributions from the positively charged
-amino group (Figure 1). Hence substantial destabilization is caused by moving the N-terminus away from a region of highly negative potential, again in accordance with experimental data (see Table I). It therefore appears that the pronounced destabilization of recombinant wild-type
-LA noted earlier is due to unfavorable chargecharge interactions.
In order to compare quantitatively the experimental data with the electrostatic calculations the mutation-induced changes in the Gibbs free energy of thermal denaturation (Gn
m) of apo- and Ca2+-loaded
-LA species were estimated from the mid-transition temperatures (tm) in Table I. The calculations were based on DSC data for native
-LA (see Figure 3), which were analyzed according to the simple two-state model followed by calculation of the free energy change upon thermal denaturation (
G) according to Equation (6), given in Materials and methods:
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The resulting Gn
m values are presented in Table I. A comparative plot of
Gn
m versus the change in the energy of chargecharge interactions (
Gq-q) is shown in Figure 4. Overall, the correlation qualitatively predicts the effects of every substitution with the possible exception of the D37N mutant. The TK-SA algorithm for this latter substitution predicted little change in stability due to changes in chargecharge interactions, whereas the experimental results showed a significant increase in stability. Since the TK-SA calculations reflect changes only in chargecharge interactions, other potential effects such as changes in hydrophobicity, hydrogen bonding, configurational entropy or secondary structure propensities may account for the additional stabilization.
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Computational analysis of chargecharge interactions in bovine -LA conducted in this work was qualitatively validated by experiment. The N-terminal sequence (residues 111) is characterized by a high proportion of negatively charged residues that cluster on the surface of the native protein. Neutralization of unfavorable chargecharge interactions in the N-terminus results in stabilization of both the apo- and Ca2+-bound protein. As also demonstrated above, an increase in thermal stability is related to an increase in calcium binding affinity. If one considers the binding of basic proteins, histones or positively charged polyamino acids, these interactions are accompanied by different effects (Permyakov et al., 2004
). Although the interaction with basic polymers increased the thermal stability of apo-
-LA, the calcium-saturated protein was destabilized (the calcium affinity was diminished also). An example that illustrates the comparable mechanism is the effect of Asp87 replacement, which directly coordinates the calcium ion in
-LA, with the neutral side chain Asn (Permyakov et al., 2001
). The resulting decrease in calcium binding affinity was about two orders of magnitude with an accompanying decrease in thermal stability of the Ca2+-loaded protein. It is likely that the same destabilization of the Ca2+-saturated form occurs in the case of
-LA interactions with the basic proteins, i.e. involvement of the highly negatively charged calcium-binding loop in the binding of basic proteins results in a decrease in calcium affinity, consequently decreasing the thermal stability of the Ca2+-loaded protein. The interaction of apo-protein with a basic polymer stabilizes
-LA via the neutralization of the highly unfavorable negative charge distribution of the calcium binding-loop (see Figure 1). Hence the N-terminus may be involved in similar compensating interactions with histones, positively charged polyamino acids and other polycations in vivo.
It should be noted that, despite many years of protein engineering studies with -LA, rarely have mutations been found that improve protein physico-chemical properties. We are aware of only two cases where a significant increase in
-LA thermal stability was found and most of these mutations were not planned via rational computational approaches (Greene et al., 1999
; Veprintsev et al., 1999
). This strategy was first proposed by Loladze et al. in 1999
(Loladze et al., 1999
) based on experimental data from several papers, which showed that mutations in surface charges can significantly alter protein stability (Grimsley et al., 1999
; Paoli et al., 1999
; Perl et al., 2000
; Spector et al., 2000
). It was further reinforced when it was shown that computational approaches are capable of qualitatively predicting the consequences of surface charge mutations on protein stability (Perl and Schmid, 2001
; Sanchez-Ruiz and Makhatadze, 2001
; Forsyth et al., 2002
; Zhou, 2002b
; Luisi et al., 2003
; Makhatadze et al., 2003
; Schwehm et al., 2003
).
The results presented here provide experimental validation for rational optimization of chargecharge interactions on a protein surface as a tool to modulate protein stability. Examination of the energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue clearly suggests that nature may have made an error with bovine -LA (as opposed to the human and most other species, which contain an N-terminal Lys!). Or was this destabilizing contribution intentional for other physiological reasons? Further studies on the differences between bovine and other
-LA species in physiological function may shed more light on this question.
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Acknowledgements |
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Received April 5, 2005; revised June 28, 2005; accepted June 30, 2005.
Edited by Gideon Schreiber
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