Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli.

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.

[1]  K. Kuwajima,et al.  Transition state in the folding of α‐lactalbumin probed by the 6‐120 disulfide bond , 1998, Protein science : a publication of the Protein Society.

[2]  L. T. Chen,et al.  Remarkable destabilization of recombinant alpha-lactalbumin by an extraneous N-terminal methionyl residue. , 1998, Protein engineering.

[3]  L. Gregoret,et al.  Stability and folding properties of a model β‐sheet protein, Escherichia coli CspA , 1998, Protein science : a publication of the Protein Society.

[4]  P. S. Kim,et al.  Hydrophobic sequence minimization of the alpha-lactalbumin molten globule. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Ueda,et al.  Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser. , 1997, Protein engineering.

[6]  Christopher M. Dobson,et al.  A residue-specific NMR view of the non-cooperative unfolding of a molten globule , 1997, Nature Structural Biology.

[7]  B. Kuhlman,et al.  Calcium binding peptides from alpha-lactalbumin: implications for protein folding and stability. , 1997, Biochemistry.

[8]  S. Anderson,et al.  Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin. , 1997, Protein engineering.

[9]  K. Kuwajima,et al.  Structural characterization of the molten globule of α‐lactalbumin by solution X‐ray scattering , 1997, Protein science : a publication of the Protein Society.

[10]  K. Kuwajima,et al.  Dominant Forces in the Recognition of a Transient Folding Intermediate of α-Lactalbumin by GroEL , 1996 .

[11]  C M Dobson,et al.  Protein Folding Monitored at Individual Residues During a Two-Dimensional NMR Experiment , 1996, Science.

[12]  L. Barron,et al.  The native-like tertiary fold in molten globule alpha-lactalbumin appears to be controlled by a continuous phase transition. , 1996, Journal of molecular biology.

[13]  P. S. Kim,et al.  Proline scanning mutagenesis of a molten globule reveals non-cooperative formation of a protein's overall topology , 1996, Nature Structural Biology.

[14]  K. Kuwajima,et al.  Rapid formation of a molten globule intermediate in refolding of α-lactalbumin , 1996 .

[15]  M. Inouye,et al.  The 19‐residue pro‐peptide of staphylococcal nuclease has a profound secretion‐enhancing ability in Escherichia coli , 1996, Molecular microbiology.

[16]  K. Brew,et al.  Crystal structures of guinea-pig, goat and bovine alpha-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase. , 1996, Structure.

[17]  S. Sugai,et al.  A synthetic peptide study on the molten globule of alpha-lactalbumin. , 1996, Journal of biochemistry.

[18]  D. Shortle.  The denatured state (the other half of the folding equation) and its role in protein stability , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  K. Kuwajima.  The molten globule state of α‐lactalbumin , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  K. Kuwajima,et al.  Effects of amino acid substitutions in the hydrophobic core of alpha-lactalbumin on the stability of the molten globule state. , 1995, Protein engineering.

[21]  R. Sauer,et al.  P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and refolding. , 1995, Biochemistry.

[22]  B. Jonsson,et al.  Folding and stability of the N-terminus of human carbonic anhydrase II. , 1995, Biochemistry.

[23]  S. Krzywda,et al.  Stability of myoglobin: a model for the folding of heme proteins. , 1994, Biochemistry.

[24]  G. Vanderheeren,et al.  Thermal unfolding of bovine alpha-lactalbumin. Comparison of circular dichroism with hydrophobicity measurements. , 1994, The Journal of biological chemistry.

[25]  Robert T. Sauer,et al.  DNA recognition by β-sheets in the Arc represser–operator crystal structure , 1994, Nature.

[26]  R. Sauer,et al.  DNA recognition by beta-sheets in the Arc repressor-operator crystal structure. , 1994, Nature.

[27]  R. Sauer,et al.  P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences , 1993, Protein science : a publication of the Protein Society.

[28]  R. L. Baldwin,et al.  Cis proline mutants of ribonuclease A. I. thermal stability , 1992, Protein science : a publication of the Protein Society.

[29]  K. Ohta,et al.  Efficient in vitro folding of the three‐disulfide derivatives of hen lysozyme in the presence of glycerol , 1992, FEBS letters.

[30]  L Serrano,et al.  The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure. , 1992, Journal of molecular biology.

[31]  K. Harata,et al.  X-ray structural evidence for a local helix-loop transition in alpha-lactalbumin. , 1992, The Journal of biological chemistry.

[32]  M. Oobatake,et al.  Hydration and heat stability effects on protein unfolding. , 1991, Progress in biophysics and molecular biology.

[33]  J. Taylor,et al.  Functional characterization of human recombinant apolipoprotein AIV produced in Escherichia coli. , 1991, European journal of biochemistry.

[34]  D. Stuart,et al.  Crystal structure of human α-lactalbumin at 1·7 Å resolution , 1991 .

[35]  T. Hynes,et al.  The crystal structure of staphylococcal nuclease refined at 1.7 Å resolution , 1991, Proteins.

[36]  F Sherman,et al.  The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation. , 1990, The Journal of biological chemistry.

[37]  I. Kumagai,et al.  Expression of goat alpha-lactalbumin in Escherichia coli and its refolding to biologically active protein. , 1990, Protein engineering.

[38]  W. Chazin,et al.  1H NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D9k. , 1989, Biochemistry.

[39]  A. Fersht,et al.  Mapping the transition state and pathway of protein folding by protein engineering , 1989, Nature.

[40]  D I Stuart,et al.  Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme. , 1989, Journal of molecular biology.

[41]  K. Kuwajima,et al.  Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin. , 1989, Journal of molecular biology.

[42]  T. Ueda,et al.  Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange. , 1987, Protein engineering.

[43]  M. Furuno,et al.  Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme. , 1987, Protein engineering.

[44]  P. Wingfield,et al.  N-terminal methionine-specific peptidase in Salmonella typhimurium. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[45]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[46]  T. Richmond,et al.  Solvent accessible surface area and excluded volume in proteins. Analytical equations for overlapping spheres and implications for the hydrophobic effect. , 1984, Journal of molecular biology.

[47]  L. Lindahl,et al.  Metal-ion-dependent hydrophobic-interaction chromatography of α-lactalbumins , 1984 .

[48]  K. Kuwajima,et al.  Intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in goat alpha-lactalbumin. , 1980, Biochimica et biophysica acta.

[49]  R L Blakeley,et al.  Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination. , 1979, Analytical biochemistry.

[50]  A. Shrake,et al.  Environment and exposure to solvent of protein atoms. Lysozyme and insulin. , 1973, Journal of molecular biology.

[51]  R. E. Webster,et al.  Protein chain initiation and deformylation in B. subtilis homogenates. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[52]  J. M. Adams,et al.  On the release of the formyl group from nascent protein. , 1968, Journal of molecular biology.

[53]  F. Sanger,et al.  N-Formyl-methionyl-S-RNA , 1964 .

[54]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[55]  V. Luzzati,et al.  Traitement statistique des erreurs dans la determination des structures cristallines , 1952 .

[56]  W. Pfeil.  Is the molten globule a third thermodynamic state of protein? The example of α‐lactalbumin , 1998 .

[57]  O. Ptitsyn,et al.  Molten globule and protein folding. , 1995, Advances in protein chemistry.

[58]  S. Sugai,et al.  Conformational comparison between α-lactalbumin and lysozyme , 1994 .

[59]  M. Inouye,et al.  Major cold shock protein of Escherichia coli. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[60]  Axel T. Brunger,et al.  Extension of molecular replacement: a new search strategy based on Patterson correlation refinement , 1990 .

[61]  K. Kuwajima,et al.  The molten globule state as a clue for understanding the folding and cooperativity of globular‐protein structure , 1989, Proteins.

[62]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[63]  K. Kuwajima,et al.  Role of the Interaction between Ionizable Groups in the Folding of Bovine α-Lactalbumin , 1981 .

[64]  C. Tanford.  Protein denaturation. , 1968, Advances in protein chemistry.