Dynamics of a monomeric insulin analogue: testing the molten-globule hypothesis.

The structure of insulin exhibits local and nonlocal differences among crystal forms and so provides an important model for analysis of protein dynamics. A novel combination of order and disorder has recently been inferred from 2D-NMR studies of the monomeric analogue des-pentapeptide(B26-B30) insulin (DPI) under acidic conditions [the molten-globule hypothesis; Hua, Q.X., Kochoyan, M., & Weiss, M.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2379-2383]. Distance-geometry structures are similar in general to crystal structures but differ by rigid-body displacements of alpha-helices; the hydrophobic core is not well ordered due to insufficient long-range restraints. To test whether such informational uncertainty may represent physical disorder, we have performed complementary studies of the thermal unfolding of DPI and its interaction with 1-anilino-8-naphthalenesulfonate (ANS). Experimental design is based on a predicted analogy between DPI and A-state models of protein-folding intermediates (the "molten globule"). Unfolding is monitored by five distinct biophysical probes: photochemical dynamic nuclear polarization (photo-CIDNP), differential scanning calorimetry (DSC), circular dichroism (CD), 1H-NMR chemical shifts, and slowly exchanging amide 1H-NMR resonances in D2O solution. The results provide evidence that DPI adopts a compact partially folded state. Because the 2D-NMR spectrum of an engineered insulin monomer under physiological conditions is similar to that of DPI under acidic conditions [Weiss, M.A., Hua, Q.X., Frank, B.H., Lynch, C., & Shoelson, S.E. (1991) Biochemistry 30, 7373-7389], we propose that the functional form of insulin is a molten globule.

[1]  L. Thim,et al.  Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation. , 2009, International journal of peptide and protein research.

[2]  S. Shoelson,et al.  Receptor binding redefined by a structural switch in a mutant human insulin , 1994, Nature.

[3]  S. M. Kristensen,et al.  Three-dimensional solution structure of an insulin dimer , 1992 .

[4]  D. Case,et al.  A new analysis of proton chemical shifts in proteins , 1991 .

[5]  R. Knegtel,et al.  The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics. , 1991, European journal of biochemistry.

[6]  H. Berendsen,et al.  Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study. , 1991, Biochemistry.

[7]  S. Shoelson,et al.  Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design. , 1991, Biochemistry.

[8]  E J Dodson,et al.  X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue. , 1991, Journal of molecular biology.

[9]  M. Weiss,et al.  Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition. , 1991, Biochemistry.

[10]  R. L. Baldwin,et al.  Probing the stability of a partly folded apomyoglobin intermediate by site-directed mutagenesis. , 1991, Biochemistry.

[11]  Timothy F. Havel,et al.  Alternating zinc fingers in the human male associated protein ZFY: 2D NMR structure of an even finger and implications for "jumping-linker" DNA recognition. , 1991, Biochemistry.

[12]  Dudley H. Williams,et al.  Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin. , 1991, Biochemistry.

[13]  S. M. Kristensen,et al.  Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure. , 1991, Journal of molecular biology.

[14]  E J Dodson,et al.  Structure of the pig insulin dimer in the cubic crystal. , 1991, Acta crystallographica. Section B, Structural science.

[15]  C. Dobson,et al.  A photochemically induced dynamic nuclear polarization study of denatured states of lysozyme. , 1991, Biochemistry.

[16]  M. Weiss,et al.  Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure. , 1990, Biochemistry.

[17]  S W Englander,et al.  Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR. , 1990, Biochemistry.

[18]  P E Wright,et al.  Structural characterization of a partly folded apomyoglobin intermediate. , 1990, Science.

[19]  R. Kaptein,et al.  Two-dimensional NMR studies on des-pentapeptide-insulin. Proton resonance assignments and secondary structure analysis. , 1990, European journal of biochemistry.

[20]  M. Roy,et al.  1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms. , 1990, The Journal of biological chemistry.

[21]  M. Karplus,et al.  Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: assignment of aromatic resonances with application to protein folding, structure, and dynamics. , 1989, Biochemistry.

[22]  J. Sturtevant,et al.  A scanning calorimetric study of the thermal denaturation of the lysozyme of phage T4 and the Arg 96----His mutant form thereof. , 1989, Biochemistry.

[23]  R. Mirmira,et al.  Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation. , 1989, The Journal of biological chemistry.

[24]  E. Dodson,et al.  Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer , 1989, Nature.

[25]  D. Laurents,et al.  A new method for determining the heat capacity change for protein folding. , 1989, Biochemistry.

[26]  A. Fink,et al.  Conformational states of beta-lactamase: molten-globule states at acidic and alkaline pH with high salt. , 1989, Biochemistry.

[27]  C M Dobson,et al.  Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin. , 1989, Biochemistry.

[28]  K Wüthrich,et al.  Determination of the complete three-dimensional structure of the alpha-amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry. , 1988, Journal of molecular biology.

[29]  P. Privalov,et al.  Thermodynamic study of the apomyoglobin structure. , 1988, Journal of molecular biology.

[30]  Timothy F. Havel,et al.  Protein structures in solution by nuclear magnetic resonance and distance geometry. The polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN. , 1987, Journal of molecular biology.

[31]  O. Ptitsyn.  Protein folding: Hypotheses and experiments , 1987 .

[32]  J. Hoch,et al.  Interpretation of ring-current shifts in proteins: application to phage λ repressor , 1987 .

[33]  V. Hruby,et al.  Design and synthesis of glucagon partial agonists and antagonists. , 1986, Biochemistry.

[34]  O. Ptitsyn,et al.  Physical nature of the phase transition in globular proteins , 1986, FEBS letters.

[35]  J. Gerlt,et al.  Thermal denaturation of staphylococcal nuclease. , 1985, Biochemistry.

[36]  O. Ptitsyn,et al.  Noncooperative temperature melting of a globular protein without specific tertiary structure: Acid form of bovine carbonic anhydrase B , 1985, Biopolymers.

[37]  L. Thim,et al.  Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21). , 1985, The Journal of biological chemistry.

[38]  T. James,et al.  A theoretical study of distance determinations from NMR. Two-dimensional nuclear overhauser effect spectra , 1984 .

[39]  Z. Dauter,et al.  Insulin's structure as a modified and monomeric molecule , 1984 .

[40]  S. Weinstein,et al.  Photochemically induced nuclear polarization study of the accessibility of tyrosines in insulin. , 1984, Biochemistry.

[41]  H. Hinz,et al.  Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters. , 1983, Journal of molecular biology.

[42]  K Wüthrich,et al.  Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance. , 1983, Journal of molecular biology.

[43]  Cyrus Chothia,et al.  Transmission of conformational change in insulin , 1983, Nature.

[44]  K. Wüthrich,et al.  Amide protein exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution. Studies with two-dimensional nuclear magnetic resonance. , 1982, Journal of molecular biology.

[45]  M. Karplus,et al.  Fluctuations and averaging of proton chemical shifts in the bovine pancreatic trypsin inhibitor. , 1982, Biochemistry.

[46]  K Wüthrich,et al.  Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Glucagon bound to perdeuterated dodecylphosphocholine micelles. , 1982, Journal of molecular biology.

[47]  K Wüthrich,et al.  Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor. , 1982, Journal of molecular biology.

[48]  O. Ptitsyn,et al.  α‐lactalbumin: compact state with fluctuating tertiary structure? , 1981, FEBS letters.

[49]  W. Pfeil.  Thermodynamics of α-lactalbumin unfolding , 1981 .

[50]  S. Biswas,et al.  Conformational dynamics of insulin in solution. Circular dichroic studies. , 1980, Biochemistry.

[51]  M. Karplus,et al.  Proton NMR studies of the association and folding of glucagon in solution , 1980, FEBS letters.

[52]  C. Matthews,et al.  Effect of single amino acid substitutions on the thermal stability of the alpha subunit of tryptophan synthase. , 1980, Biochemistry.

[53]  D. Hodgkin,et al.  Structural relationships in the two-zinc insulin hexamer. , 1979, Canadian journal of biochemistry.

[54]  R. Sauer,et al.  The lambda repressor contains two domains. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[55]  C Boesch,et al.  1H nuclear-magnetic-resonance studies of the molecular conformation of monomeric glucagon in aqueous solution. , 1978, European journal of biochemistry.

[56]  E. Freire,et al.  Statistical mechanical deconvolution of thermal transitions in macromolecules. I. Theory and application to homogeneous systems , 1978 .

[57]  K. Kuwajima,et al.  Three-state denaturation of α-lactalbumin by guanidine hydrochloride , 1976 .

[58]  M. Karplus,et al.  Protein-folding dynamics , 1976, Nature.

[59]  T. Blundell,et al.  Receptor-binding region of insulin , 1976, Nature.

[60]  Ian J. Tickle,et al.  X-ray analysis of glucagon and its relationship to receptor binding , 1975, Nature.

[61]  J. Deisenhofer,et al.  Crystallographic refinement of the structure of bovine pancreatic trypsin inhibitor at l.5 Å resolution , 1975 .

[62]  P. Privalov,et al.  A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. , 1974, Journal of molecular biology.

[63]  E. Baker,et al.  Structure of Rhombohedral 2 Zinc Insulin Crystals , 1969, Nature.

[64]  G. Fasman,et al.  A fluorescent probe, toluidinyl-naphthalene-sulfonate, specific for the β structure of poly-l-lysine☆ , 1968 .

[65]  L. Stryer,et al.  The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. , 1965, Journal of molecular biology.

[66]  K Wüthrich,et al.  Determination of three-dimensional protein structures in solution by nuclear magnetic resonance: an overview. , 1989, Methods in enzymology.

[67]  P. S. Kim,et al.  Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. , 1982, Annual review of biochemistry.

[68]  H. Gattner.  B-Kettenverkürzung von polymergebundenem Insulin mit Pepsin, I. Darstellung und Eigenschaften von Des-Pentapeptid (B26-30)-Rinderinsulin , 1975 .

[69]  E. Bradbury,et al.  A conformational study of glucagon. , 1968, European journal of biochemistry.