1 Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia, 2 Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, 3 Departments of Veterinary Biosciences and Biochemistry and 4 Departments of Chemistry and Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA
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Abstract |
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Keywords: -lactalbumin/calcium binding/fluorescence/thermal stability
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Introduction |
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Although the process of Ca(II) binding by -LA is well characterized both structurally and quantitatively (Permyakov et al., 1981
; Griko and Remeta, 1999
), the role of the process in lactose synthesis is still far from clear (Permyakov and Berliner, 2000
). Nevertheless, the binding of Ca(II) to
-LA is necessary for correct folding of the protein (Rao and Brew, 1989
), which emphasizes a significance of the Ca(II)-binding loop of
-LA for its folding.
In order to evaluate contributions of each of the Ca(II)-co-ordinating carboxylic charges into the process of the Ca(II) binding by bovine -LA, we previously prepared a set of
-LA mutants with ligating Asp residues substituted by Ala (Anderson et al., 1997
). Measurements of Ca(II) affinity of these mutants revealed that D87A and D88A were unable to bind calcium ions, which we interpreted to mean that residues Asp87 and Asp88 are required for effective Ca(II) binding. Both mutants exhibited a total loss of tertiary structure with a partial loss of secondary structure. The structural rearrangement could be caused by transition to a molten globule state or by incorrect folding of the mutant
-LAs, caused by their decreased calcium affinity as predicted by the work of Rao and Brew (Rao and Brew, 1989
). In any case, the observed distortion of
-LA structure, induced by alanine substitution, makes accurate measurement of calcium affinity difficult and prevents the delineation of the calcium site determinants.
In order to minimize structural distortion in mutant bovine -LAs, we substituted Asp87 with Asn. Asparagine lacks the non-compensated negative charge of a carboxylate group, yet has the same side-chain volume and geometry. Studies of the physico-chemical properties of the resulting D87N
-LA by means of intrinsic protein fluorescence and circular dichroism (CD) spectroscopy allowed us to measure both the electrostatic and geometric contributions of the side chain for calcium binding.
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Materials and methods |
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Bovine -lactalbumin (lot 128F-8140) and Aeromonas proteolitica aminopeptidase (E.C. 3.4.11.10, lot 27H4050), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) and ethylene glycol-bis(ß-aminoethyl ether)-N,N,N ',N '-tetraacetic acid (EGTA) were purchased from Sigma Chemical Co. (St Louis, MO). All other chemicals were reagent grade or better. Solutions were prepared from double-distilled, demineralized water. Protein concentrations were evaluated spectrophotometrically using an extinction coefficient, E1%280 nm = 20.1 (Kronman and Andreotti, 1964
).
Preparation of recombinant -lactalbumins
Recombinant proteins D87A and D87N-LA were expressed in E.coli as previously described (Anderson et al., 1997
; Peterson et al., 1999
). As a consequence of protein expression the recombinant protein contained an extra methionine residue at the N-terminus, which is known to destabilize
-LA structure, thermal stability and calcium affinity (Ishikawa et al., 1998
; Chaudhuri et al., 1999
; Veprintsev et al., 1999
). In order to eliminate this perturbation the N-terminal Met was removed by A.proteolitica aminopeptidase enzyme (Wilkes et al., 1973
; Prescott and Wilkes, 1976
) treatment as described in our earlier work (Veprintsev et al., 1999
). The efficacy and precision of this procedure were confirmed by mass spectrometry and CD.
Fluorescence and CD spectroscopy
Fluorescence measurements were performed on a Perkin-Elmer LS-50B spectrofluorimeter. All spectra were corrected and fitted to log-normal curves (Burstein and Emelyanenko, 1996) using non-linear regression analysis (Marquardt, 1963
). The maximum positions of the spectra were obtained from the fitted data. Protein fluorescence was excited at 280 nm. 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 denatured state was calculated from the plots of the temperature dependence of emission intensity at a fixed wavelength as previously described (Permyakov and Burstein, 1984
). Calcium-binding affinity was measured using a spectrofluorimetric CaCl2/EGTA-titration method described earlier (Permyakov et al., 1981
). The accuracy of the determination of the calcium-binding constants was approximately half an order of magnitude. Estimation of parameters of theoretical models was carried out according to Marquardt (Marquardt, 1963
). Protein concentrations were between 2 and 6 µM.
CD measurements were performed on a AVIV 62DS or Jasco J-500A spectropolarimeter. Typical instrument conditions were: scan rate, 5 nm/min; time constant, 8 s. The pathlength was 0.1 mm for the far-UV studies and 5 mm for the near-UV region. Protein concentrations were in the 25 to 100 µM range.
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Results and discussion |
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Figure 1A and B depict the near- and far-CD spectra, respectively, of apo- and Ca(II)-saturated native
-LA, D87A
-LA, D87N
-LA, and des-Met D87N
-LA at 5°C (10 mM HEPES or Tris, pH 8.0). Figure 1A
demonstrates the expected recovery of tertiary structure in D87N upon the enzymatic removal of the N-terminal methionine yielding desMetD87N
-LA. This was the case for both apo- and Ca(II)-saturated desMet D87N
-LA.
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Thermal denaturation of desMetD87N -LA
Figure 2A and B compare fluorescence thermal denaturation curves for the apo- and calcium-bound forms of desMetD87N
-LA and native
-LA, respectively. Note that the fractional conversion data were obtained from changes in fluorescence emission intensity at a fixed wavelength after Permyakov and Burstein (Permyakov and Burstein, 1984
). It is clear that apo-desMet D87N
-LA is ~7°C more stable than native apo-
-LA (Figure 2B
). In addition, apo-desMetD87N
-LA is ~6 nm red-shifted (Figure 2A
) compared with the native protein, which indicates a more mobile and/or more polar tryptophan environment. The same conclusions were drawn from the near-UV CD data at 5°C (Figure 1A
), where the ellipticity at 270 nm for apo-desMetD87N was reduced by ~32% compared to apo-native
-LA, which suggests a partial loss of tertiary structure in desMetD87N
-LA. At the same time, the secondary structures of both forms are identical as judged from far-UV CD spectra (Figure 1B
).
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Calcium affinity of desMetD87N -LA
The calcium-binding affinity of desMetD87N -LA was measured by direct titration with calcium chloride as monitored by changes in intrinsic tryptophan fluorescence at 40°C (10 mM HEPES, pH 8.0) which is above the thermal transitions for both apo-forms and below the thermal transitions for the Ca(II)-bound forms (see Figure 2
). This temperature was chosen because it offered the most sensitive spectral changes between the apo- and calcium-bound forms allowing the most accurate determinations of calcium affinity. The experimental points were fit to a simple one-site binding scheme:
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In contrast to the results with D87A -LA, where our CD measurements revealed the total disorganization of tertiary structure, a major loss of secondary structure, the inability to bind calcium and a loss of a co-operative thermal transition (Anderson et al., 1997
), desMet D87N
-LA retains each of these structural and functional features. The failure of D87A
-LA to fold into a functional form, as reflected in negligible calcium affinity, demonstrates the necessity of calcium binding for correct folding and disulfide bond formation as pointed out earlier by Rao and Brew (Rao and Brew, 1989
) and Chandra et al. (Chandra et al., 1998
).
An unexpected property of desMetD87N -LA was a substantial increase in the thermal stability of the apo-form (~7°C), despite a moderate decrease in tertiary structural rigidity (~32% decrease in molar ellipticity at 270 nm). This observation is consistent with the differential scanning calorimetry studies of Griko and Remeta (Griko and Remeta, 1999
), who suggested that the stability of apo-
-LA was significantly reduced due to negative chargecharge interactions in the calcium-binding site. Hence, virtually any substitution of one of these residues by a non-anionic side chain should increase the stability of the apo-form.
In summary, the residue change from Asp to Asn was absolutely minimal from both packing volume and ligand co-ordination considerations. The Asn side chain still allows for calcium co-ordination via the same carbonyl oxygen that exists in the native protein. The only significant changes were the removal of a negative charge and the potential loss of an alternative oxygen atom for ligand co-ordination. Since the metal-free native protein is characterized by five charged carboxylates (Asp 82, -83, -84, -87 and -88) in the calcium-binding loop region, three of which directly co-ordinate the cation and one of which is not that distant), it is clear that electrostatic repulsion is a key factor in folding and stabilization of the apo- and calcium-bound forms, respectively. This was supported by recent X-ray structures of bovine -LA which clearly displayed a retreat of Asp87 from the other liganding side chains, as well as the loss of the two calcium co-ordinating waters in the apo-form (Chrysina et al., 2000
). Consequently, electrostatic repulsion would appear to be a major driving force in the movement of Asp87 away from the vacant calcium co-ordinating site the apo-protein and the loss of the two key structural water molecules, initially noted in the X-ray structure of human
-LA (Acharya et al., 1991
), that bridges key Asp residues in the calcium-binding loop. One water molecule forms potential hydrogen bonds to Asp82 and Asp87. The second water bridges Asp84 and Asp87. Hence, the charge compensation afforded by either the Asp to Asn mutation in the apo-form or by Ca(II) binding in the native protein would appear to predict an increase in thermal stability. In addition, we cannot rule out the contribution of additional hydrogen bonding donor capacity resulting from the introduction of an Asn residue, which could potentially contribute additional stability. Furthermore, overall protein stability is also related to the stability of the unfolded state which this latter mutation could potentially influence. The consideration of side-chain volume is also potentially important when choosing amino acid substitution in protein engineering. The respective average volume of buried residues for the amino acids under consideration here are: Ala, 92 Å3; Asp, 125 Å3; Asn 125 Å3 (Chothia, 1975
). Initially it might seem difficult to assess quantitatively the degree to which a 26% relative void in packing volume between Ala and Asn impacts on protein structure and stability at a single site. However, residue volume cannot be overlooked as a real contribution to the remarkable differences in stability observed between the Ala and Asn mutants, since D87A
-LA was unable either to fold properly or bind calcium. Furthermore, desMet D87N
-LA resembles most other structural and functional features of the native protein, while eliminating one repulsive negative charge, yet retaining the ability to pack and hydrogen bond with other residues in the vicinity. These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice for gaining a mechanistic understanding of protein function. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.
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Notes |
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6 To whom correspondence should be addressed. E-mail: berliner{at}du.edu
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Acknowledgments |
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References |
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Received February 13, 2001; revised June 28, 2001; accepted July 16, 2001.