FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2088751510> ?p ?o ?g. }

• W2088751510 endingPage "47661" @default.
• W2088751510 startingPage "47653" @default.
• W2088751510 abstract "Mitogen-activated protein (MAP) kinase-mediated phosphorylation of specific residues in tyrosine hydroxylase leads to an increase in enzyme activity. However, the mechanism whereby phosphorylation affects enzyme turnover is not well understood. We used a combination of fluorescence resonance energy transfer (FRET) measurements and molecular dynamics simulations to explore the conformational free energy landscape of a 10-residue MAP kinase substrate found near the N terminus of the enzyme. This region is believed to be part of an autoregulatory sequence that overlies the active site of the enzyme. FRET was used to measure the effect of phosphorylation on the ensemble of peptide conformations, and molecular dynamics simulations generated free energy profiles for both the unphosphorylated and phosphorylated peptides. We demonstrate how FRET transfer efficiencies can be calculated from molecular dynamics simulations. For both the unphosphorylated and phosphorylated peptides, the calculated FRET efficiencies are in excellent agreement with the experimentally determined values. Moreover, the FRET measurements and molecular simulations suggest that phosphorylation causes the peptide backbone to change direction and fold into a compact structure relative to the unphosphorylated state. These results are consistent with a model of enzyme activation where phosphorylation of the MAP kinase substrate causes the N-terminal region to adopt a compact structure away from the active site. The methods we employ provide a general framework for analyzing the accessible conformational states of peptides and small molecules. Therefore, they are expected to be applicable to a variety of different systems. Mitogen-activated protein (MAP) kinase-mediated phosphorylation of specific residues in tyrosine hydroxylase leads to an increase in enzyme activity. However, the mechanism whereby phosphorylation affects enzyme turnover is not well understood. We used a combination of fluorescence resonance energy transfer (FRET) measurements and molecular dynamics simulations to explore the conformational free energy landscape of a 10-residue MAP kinase substrate found near the N terminus of the enzyme. This region is believed to be part of an autoregulatory sequence that overlies the active site of the enzyme. FRET was used to measure the effect of phosphorylation on the ensemble of peptide conformations, and molecular dynamics simulations generated free energy profiles for both the unphosphorylated and phosphorylated peptides. We demonstrate how FRET transfer efficiencies can be calculated from molecular dynamics simulations. For both the unphosphorylated and phosphorylated peptides, the calculated FRET efficiencies are in excellent agreement with the experimentally determined values. Moreover, the FRET measurements and molecular simulations suggest that phosphorylation causes the peptide backbone to change direction and fold into a compact structure relative to the unphosphorylated state. These results are consistent with a model of enzyme activation where phosphorylation of the MAP kinase substrate causes the N-terminal region to adopt a compact structure away from the active site. The methods we employ provide a general framework for analyzing the accessible conformational states of peptides and small molecules. Therefore, they are expected to be applicable to a variety of different systems. Phosphorylation of specific amino acids near the surface of a protein is an almost universal mechanism of protein activation that has been long appreciated (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar, 2Cohen P. Trends Biochem. Sci. 2000; 25: 596-601Google Scholar). Yet the mechanism whereby phosphorylation modifies the activity of the protein is not well understood (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar). Analysis of phosphorylation sites from different enzymes reveals common themes. In eukaryotes, phosphorylation typically occurs at tyrosine, threonine, or serine side chains, and these phosphorylated residues often form a network of hydrogen bonds with adjacent positively charged arginine residues (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar, 3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar, 4Johnson L. FASEB J. 1992; 6: 2274-2282Google Scholar, 5Russo A. Jeffrey P. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Google Scholar, 6Feher V.A. Yih-Ling T. Hoch J.A. Cavanagh J. FEBS Lett. 1998; 425: 1-6Google Scholar). The network of hydrogen bonds and salt bridges that form can then communicate phosphorylation to distant areas of the protein (6Feher V.A. Yih-Ling T. Hoch J.A. Cavanagh J. FEBS Lett. 1998; 425: 1-6Google Scholar). In the case of yeast glycogen phosphorylase, phosphorylation occurs at a threonine residue located near the N terminus, a region that overlies the active site of the enzyme (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). The enzyme, which normally exists as a homodimer, contains two active catalytic sites, one in each monomer (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). Phosphorylation at this site causes the N-terminal region to fold into a compact structure that wedges between the dimer interface. This structural change helps to reorient the active site in a manner that facilitates enzymatic activation (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). Examples such as this suggest that large scale movements of flexible regions within a protein are an important component of phosphorylation-induced protein activation (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar, 4Johnson L. FASEB J. 1992; 6: 2274-2282Google Scholar).The aromatic amino acid hydroxylase superfamily comprises a set of enzymes that require phosphorylation at specific sites to become activated (7Fitzpatrick P. Annu. Rev. Biochem. 1999; 68: 355-381Google Scholar, 8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar, 9Jiang G. Yohrling G. Schmitt J. Vrana K. J. Mol. Biol. 2000; 302: 1005-1017Google Scholar). The three enzymes within this class, tryptophan hydroxylase, phenylalanine hydroxylase, and tyrosine hydroxylase, share significant sequence homology within their catalytic domains and are expected to have similar tertiary structures in this region (8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar, 10Vrana K. Nat. Struct. Biol. 1999; 6: 401-402Google Scholar). Unfortunately, complete x-ray crystallographic identification of the structure of all three enzymes has been limited as these compounds are particularly difficult to purify and crystallize (8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar, 9Jiang G. Yohrling G. Schmitt J. Vrana K. J. Mol. Biol. 2000; 302: 1005-1017Google Scholar). The structure of one member of this superfamily, phenylalanine hydroxylase, was recently presented in both the phosphorylated and unphosphorylated state (8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar). The structure is composed of an N-terminal regulatory domain and a C-terminal catalytic domain (10Vrana K. Nat. Struct. Biol. 1999; 6: 401-402Google Scholar). The N-terminal domain contains a 15-residue sequence (residues 19–33) that extends across the active site in the catalytic domain. However, phenylalanine hydroxylase is phosphorylated at Ser-16, and unfortunately, the first 18 residues in the structure were not identified in the electron density in either the phosphorylated or the unphosphorylated structures (8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar). This suggests that this region does not have a well defined three-dimensional structure in the absence of the substrate, phenylalanine. As a result, the two structures are quite similar. Because the structure of the phosphorylated region remains unknown, the structures of the C-terminal domains do not immediately yield information on the mechanism of phosphorylation-induced enzyme activation.Tyrosine hydroxylase, another member of this superfamily, catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine, which are molecules involved in the regulation of heart rate, blood pressure, and behavior (11Wevers R.A. Andel J. Brautigam C. Geurtz B. Heuvel J. Steenbergen-Spanjers G. Smeitink J. Hoffman G. Gabreels F. J. Inherited Metab. Dis. 1999; 22: 364-373Google Scholar). The structure of the C-terminal catalytic domain has also been solved recently and bears remarkable similarity to the corresponding domain from phenylalanine hydroxylase (10Vrana K. Nat. Struct. Biol. 1999; 6: 401-402Google Scholar, 12Goodwill K. Sabatier C. Marks C. Raag R. Fitzpatrick P. Stevens R. Nat. Struct. Biol. 1997; 4: 578-585Google Scholar). However, no depiction of the structure of the regulatory domain currently exists. Because all of the phosphorylation sites of the protein are located within the regulatory domain, it is difficult to determine how phosphorylation affects the activity of the enzyme based on an analysis of the catalytic domain alone. In situ, tyrosine hydroxylase is phosphorylated at four distinct serine residues, all located in the N-terminal autoregulatory region (Ser-8, Ser-19, Ser-31, and Ser-40) (13Haycock J. Ahn N. Cobb M. Krebs E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Google Scholar, 14Haycock J. J. Biol. Chem. 1990; 265: 11682-11691Google Scholar). However, only phosphorylation of Ser-19, Ser-31, and Ser-40 has been associated with an increase in enzyme activity (14Haycock J. J. Biol. Chem. 1990; 265: 11682-11691Google Scholar, 15Sutherland C. Alterio J. Campbell D. Le Bourdelles B. Mallet J. Haavik J. Cohen P. Eur. J. Biochem. 1993; 217: 715-722Google Scholar). Phosphorylation of Ser-40 results in an increase in activity even in the presence of known feedback inhibitors (16Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Google Scholar). By contrast, recent data (17Bevilaqua L. Graham M.E. Dunkley P.R. Nagy-Felsobuki E. Dickson P.W. J. Biol. Chem. 2001; 276: 40411-40416Google Scholar) suggest that phosphorylation of Ser-19 affects the activity of tyrosine hydroxylase by facilitating the phosphorylation of Ser-40,i.e. the rate constant for Ser-40 phosphorylation is ∼2–3-fold higher when Ser-19 is phosphorylated first. It has been suggested that upon phosphorylation at Ser-19, tyrosine hydroxylase adopts a more open conformation that enables kinases to have greater access to Ser-40 (17Bevilaqua L. Graham M.E. Dunkley P.R. Nagy-Felsobuki E. Dickson P.W. J. Biol. Chem. 2001; 276: 40411-40416Google Scholar).Phosphorylation of Ser-19 and Ser-40 are mediated by calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, respectively (13Haycock J. Ahn N. Cobb M. Krebs E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Google Scholar, 14Haycock J. J. Biol. Chem. 1990; 265: 11682-11691Google Scholar). The phosphorylation of Ser-31 is unique in that it is accomplished by the mitogen-activated protein (MAP) 1The abbreviations used are: MAP, mitogen-activated protein; FRET, fluorescence resonance energy transfer; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl 1The abbreviations used are: MAP, mitogen-activated protein; FRET, fluorescence resonance energy transfer; dansyl, 5-dimethylaminonaphthalene-1-sulfonylkinases, ERK1 and ERK2 (13Haycock J. Ahn N. Cobb M. Krebs E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Google Scholar, 18Bobrovskaya L. Odell A. Leal R.B. Dunkley P.R. J. Neurochem. 2001; 78: 490-498Google Scholar, 19Lindgren N. Goiny M. Herrera-Marschitz M. Haycock J.W. Hokfelt T. Fisone G. Eur. J. Neurosci. 2002; 15: 769-773Google Scholar). Although MAP kinase-mediated phosphorylation of Ser-31 is associated with a modest 2-fold increase in enzyme activity, phosphorylation at this site appears to be a physiologically important mechanism for the stimulation of catecholamine production in a variety of different tissues (18Bobrovskaya L. Odell A. Leal R.B. Dunkley P.R. J. Neurochem. 2001; 78: 490-498Google Scholar, 19Lindgren N. Goiny M. Herrera-Marschitz M. Haycock J.W. Hokfelt T. Fisone G. Eur. J. Neurosci. 2002; 15: 769-773Google Scholar, 20Mitchell J. Hardie D. Vulliet P. J. Biol. Chem. 1990; 36: 22358-22364Google Scholar). Therefore, deciphering the structural basis of enzyme activation by phosphorylation of Ser-31 is of particular interest.Haycock et al. (13Haycock J. Ahn N. Cobb M. Krebs E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Google Scholar) have identified a 10-residue peptide from tyrosine hydroxylase that contains the Ser-31 phosphorylation site, i.e. residues 24–33. An analysis of the structure of phenylalanine hydroxylase and its sequence alignment to tyrosine hydroxylase suggests that this peptide sequence forms a flexible region that overlies the catalytic site (13Haycock J. Ahn N. Cobb M. Krebs E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Google Scholar). Because the structure of the regulatory domain of tyrosine hydroxylase is not known, we explored the structural consequences of phosphorylating this peptide to gain insight into the mechanism of MAP kinase-induced enzyme activation. Comparison with the homologous phenylalanine structure suggests that this peptide forms a disordered region that does not form stable contacts with any other residues within the protein (8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar). Therefore, an analysis of the accessible conformations of the peptide alone may yield insights into how phosphorylation of this region affects the structure of the protein.In this work we employed a combination of fluorescent resonance energy transfer (FRET) (21Selvin P.R. Nat. Struct. Biol. 2000; 7: 730-734Google Scholar, 22Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983Google Scholar) experiments and molecular dynamics simulations (25Brooks C. Karplus M. Pettitt B.M. Proteins, A Theoretical Perspective of Dynamics, Structure, and Thermodynamics. John Wiley & Sons, Inc., New York1988Google Scholar) to determine how phosphorylation affects the accessible conformational states of the peptide. Such an approach has broad appeal because it allows one to obtain the equilibrium distribution of states using FRET without the need to compute and analyze donor decay profiles (26Levin A.D. A FRET-based and Molecular Dynamics Study of Phosphage-induced Conformational Changes in Oligopeptides.M.Sc. thesis. MIT Press, Cambridge, MA2002Google Scholar). Furthermore, the two methods are complementary and yield a wealth of information on the equilibrium distribution of conformations for this biologically relevant peptide.DISCUSSIONThe regulation of cellular function is a complex process. Reversible phosphorylation of specific sites in a given protein is an often-utilized mechanism for short term protein activation and/or deactivation (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar). As such, phosphorylation can be thought of as a molecular switch that modifies the function of a protein for as long as the protein remains phosphorylated. Although the mechanism whereby phosphorylation results in protein activation has been elucidated in only a handful of proteins (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar, 3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar, 4Johnson L. FASEB J. 1992; 6: 2274-2282Google Scholar, 6Feher V.A. Yih-Ling T. Hoch J.A. Cavanagh J. FEBS Lett. 1998; 425: 1-6Google Scholar), it is likely that phosphorylation mediates protein activation by inducing important conformational changes within the tertiary structure of the protein. Such structural modifications can be achieved in a variety of different ways, and one common mechanism involves the phosphorylation of a site located within a flexible, solvent-exposed region of the protein (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar,4Johnson L. FASEB J. 1992; 6: 2274-2282Google Scholar).In this work, changes in the emission spectra of the peptide of interest upon phosphorylation were determined using FRET experiments. These data were supplemented with results obtained from detailed molecular dynamics simulations to determine how phosphorylation affects the accessible conformational states of the peptide. A great body of literature exists on applying FRET to rigid molecules that have well defined structures in solution (22Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983Google Scholar). By contrast, in this work we apply FRET to small peptides that can adopt a number of different conformations in aqueous solvent (25Brooks C. Karplus M. Pettitt B.M. Proteins, A Theoretical Perspective of Dynamics, Structure, and Thermodynamics. John Wiley & Sons, Inc., New York1988Google Scholar). Typically, FRET is used in conjunction with time-resolved donor decay measurements to obtain the distribution of distances between a given donor and acceptor within a flexible macromolecule (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar, 33Gao J. Yao J. Squier T. Biophys. J. 2001; 80: 1791-1801Google Scholar, 34Tezcan A. Findley W. Crane B.R. Ross S.A. Lyubovitsky J.G. Gray H.B. Winkler J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8626-8630Google Scholar, 35Imperali B. Rickert K.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 97-101Google Scholar). This is possible because time-resolved decay spectra are a function, in part, of the donor-acceptor distance distribution (22Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983Google Scholar). However, the interpretation of these spectra is complicated by the fact that different conformational states relax at different rates. Because FRET measures the rate of energy transfer between an energy donor and acceptor that are in relatively close proximity of one another, conformations with small donor-acceptor distances relax at a fast rate relative to conformations with large donor-acceptor distances (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar). This introduces some uncertainty into measurements of the distance distribution because spectroscopic decay experiments record spectra on time scales that are longer than the average decay time of the sample. As a result, only conformations with large donor-acceptor distances will contribute to points on the emission spectra that are obtained at later times (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar), creating a phenomenon that is equivalent to a spectroscopic illusion. Although the equilibrium distribution of conformations does not change, at later time points it appears as if only the states with long donor-acceptor distances are present since these conformations are the only ones that continue to contribute to the decay emission spectra (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar). Therefore, the spectroscopically observed distribution of conformations changes with time although the true equilibrium distribution does not.In previous studies (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar, 35Imperali B. Rickert K.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 97-101Google Scholar), this artifact of time-dependent FRET measurements has been accounted for by modeling the spectroscopically observed change in the distribution of states as a diffusive process. However, using this method to obtain the equilibrium distribution of states is problematic because it requires the numerical solution of a complex differential equation (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar, 35Imperali B. Rickert K.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 97-101Google Scholar). Given the difficulty in obtaining accurate numerical solutions, one must assume a specific form(s) of the probability distribution of states (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar). By contrast, in this work we employ detailed molecular simulations to derive the distribution of conformational states for the peptides of interest, and we compare these data to the experimentally obtained FRET transfer efficiencies. In addition, we demonstrate how transfer efficiencies can be calculated from the equilibrium distribution of states or, equivalently, the free energy profile. If the calculated transfer efficiency agrees with the experimental result, as in this case, we assume that the calculated potential of mean force is an accurate representation of the free energy landscape of the peptide. In this work, both the fluorescent resonance experiments and the molecular dynamics simulations suggest that phosphorylation induces a dramatic change in the equilibrium distribution of conformational states. The combination of these techniques not only adds to the understanding of this family of enzymes but also may be extended to other as yet undefined problems in structural biochemistry.One limitation of the approach is that it is most useful when the data from the simulations agrees with the spectroscopic measurements. Comparison of FRET emission spectra with data derived from molecular simulations provides a mechanism for the validation of dynamic simulations performed on the system of interest. If the spectroscopic measurements are correct, then discrepancies between the calculated and measured FRET efficiencies can be used to make improvements in the simulation methodology and force field. Conversely, shortcomings in the experimental protocol for measuring FRET efficiencies may be responsible for differences between the calculated and measured result. Discrepancies between the two values should prompt a reexamination of both the experimental protocol and the simulation methodology.In this work, the calculated FRET transfer efficiencies were in excellent agreement with the experimentally determined values. Moreover, the structures obtained from the molecular simulations provide a window into the detailed molecular interactions that are responsible for the shift in the emission spectra. In the unphosphorylated state, a salt bridge forms between the side chain of Arg-11 and Asp-5. At closer end-to-end distances, additional electrostatic interactions are formed that are entropically unfavorable relative to the lowest energy state. By contrast, in the phosphorylated state, the Arg-11 side chain hydrogen-bonds to the side chain of the phosphorylated serine residue in a manner that is similar to what has been observed in other systems (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). In its new position, the Arg side chain is removed from its original orientation, and this allows other charged residues to form additional salt bridges. These interactions result in dramatic changes in the conformational free energy landscape of the peptide.An examination of the main chain conformation from both peptides suggests that phosphorylation could result in a dramatic change in the direction of the path of the N-terminal autoregulatory sequence. This change in direction could affect protein activation in a number of ways. First and foremost, since the N-terminal domain is believed to lie over the catalytic site, the new direction of the N-terminal region may remove steric hindrances that would prevent the substrate from entering the catalytic site. Second, since an additional tyrosine hydroxylase phosphorylation site, Ser-19, is located within the proximal portion of the N-terminal regulatory domain, this conformational change may help to reorient this residue so that it can interact with other residues near the surface of the protein. Prior work suggests that phosphorylation of Ser-19 promotes phosphorylation of Ser-40 (17Bevilaqua L. Graham M.E. Dunkley P.R. Nagy-Felsobuki E. Dickson P.W. J. Biol. Chem. 2001; 276: 40411-40416Google Scholar). This raises the interesting possibility that phosphorylation of Ser-31 may actually facilitate the interaction between Ser-19 and Ser-40. This notion is consistent with recent work (19Lindgren N. Goiny M. Herrera-Marschitz M. Haycock J.W. Hokfelt T. Fisone G. Eur. J. Neurosci. 2002; 15: 769-773Google Scholar) that suggests that inhibiting MAP kinase-induced phosphorylation of Ser-31 also leads to an increase in Ser-40 phosphorylation.Because the structure of the N-terminal domain of tyrosine hydroxylase is not known, the orientations shown in Fig. 5 remain speculative. We cannot rule out that the N-terminal regulatory domain makes interactions with other residues within the protein in the phosphorylated state. However, the fact that the phosphorylated N-terminal region in the related protein, phenylalanine hydroxylase, is disordered in both the unphosphorylated and phosphorylated structures suggests that, in the absence of the substrate, the phosphorylated autoregulatory region does not interact with any other residues in the molecule. Another possibility is that the phosphorylated autoregulatory region interacts with the substrate, and as a result, this region adopts an unexpected main chain conformation in the phosphorylated state. In this scenario, activation would be a two-step process that involves both phosphorylation of specific sites and the binding of tyrosine to the active site. Similar two-step mechanisms have been proposed for phenylalanine hydroxylase as well as other systems (8Kobe B. Jennings I. House C. Michell B. Goodwill K. Santarsiero B. Stevens R. Cotton R. Kemp B. Nat. Struct. Biol. 1999; 6: 442-448Google Scholar,36Stevenson L. Deal M. Hagopian J. Lew J. Biochemistry. 2002; 41: 8528-8534Google Scholar). Nevertheless, the work presented here forms a viable, and testable, model for the structural consequences for MAP kinase-mediated phosphorylation of Ser-31.Both FRET and molecular dynamics simulations are methods that can be used to probe the free energy surface of molecules. In this study, we demonstrated how the two complementary methods could be used to study significant conformational changes in a biologically relevant peptide. These data are used to derive the equilibrium distribution of conformations. Unlike prior methods, this distribution is obtained without measuring donor decay profiles (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar). Furthermore, no assumptions about the particular form of the distribution of states are needed to obtain the free energy profile (32Beechem J. Haas E. Biophys. J. 1989; 55: 1225-1236Google Scholar). Overall, the method presented in this work is a potentially powerful approach for performing detailed conformational analyses on peptides and small molecules of biological interest. Phosphorylation of specific amino acids near the surface of a protein is an almost universal mechanism of protein activation that has been long appreciated (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar, 2Cohen P. Trends Biochem. Sci. 2000; 25: 596-601Google Scholar). Yet the mechanism whereby phosphorylation modifies the activity of the protein is not well understood (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar). Analysis of phosphorylation sites from different enzymes reveals common themes. In eukaryotes, phosphorylation typically occurs at tyrosine, threonine, or serine side chains, and these phosphorylated residues often form a network of hydrogen bonds with adjacent positively charged arginine residues (1Graves J.D. Krebs E.G. Pharmacol. Ther. 1999; 82: 111-121Google Scholar, 3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar, 4Johnson L. FASEB J. 1992; 6: 2274-2282Google Scholar, 5Russo A. Jeffrey P. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Google Scholar, 6Feher V.A. Yih-Ling T. Hoch J.A. Cavanagh J. FEBS Lett. 1998; 425: 1-6Google Scholar). The network of hydrogen bonds and salt bridges that form can then communicate phosphorylation to distant areas of the protein (6Feher V.A. Yih-Ling T. Hoch J.A. Cavanagh J. FEBS Lett. 1998; 425: 1-6Google Scholar). In the case of yeast glycogen phosphorylase, phosphorylation occurs at a threonine residue located near the N terminus, a region that overlies the active site of the enzyme (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). The enzyme, which normally exists as a homodimer, contains two active catalytic sites, one in each monomer (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). Phosphorylation at this site causes the N-terminal region to fold into a compact structure that wedges between the dimer interface. This structural change helps to reorient the active site in a manner that facilitates enzymatic activation (3Lin K. Rath V. Dai S. Fletterick R. Hwang P. Science. 1996; 273: 1539-1541Google Scholar). Examples such as this suggest that large scale movements of flexible regions within a protei" @default.
• W2088751510 created "2016-06-24" @default.
• W2088751510 creator A5005931821 @default.
• W2088751510 creator A5017251809 @default.
• W2088751510 creator A5024941370 @default.
• W2088751510 date "2002-12-01" @default.
• W2088751510 modified "2023-10-10" @default.
• W2088751510 title "Phosphorylation-induced Conformational Changes in a Mitogen-activated Protein Kinase Substrate" @default.
• W2088751510 cites W133369315 @default.
• W2088751510 cites W1490295882 @default.
• W2088751510 cites W1496943120 @default.
• W2088751510 cites W1505755593 @default.
• W2088751510 cites W1512092650 @default.
• W2088751510 cites W1543186331 @default.
• W2088751510 cites W1565308849 @default.
• W2088751510 cites W1821566974 @default.
• W2088751510 cites W1868339174 @default.
• W2088751510 cites W1966719900 @default.
• W2088751510 cites W1969163423 @default.
• W2088751510 cites W1969345506 @default.
• W2088751510 cites W1969842022 @default.
• W2088751510 cites W1988758124 @default.
• W2088751510 cites W1990783034 @default.
• W2088751510 cites W1994172525 @default.
• W2088751510 cites W1997835441 @default.
• W2088751510 cites W2000954692 @default.
• W2088751510 cites W2011590971 @default.
• W2088751510 cites W2019373541 @default.
• W2088751510 cites W2029790723 @default.
• W2088751510 cites W2046036275 @default.
• W2088751510 cites W2064514012 @default.
• W2088751510 cites W2068924803 @default.
• W2088751510 cites W2073558017 @default.
• W2088751510 cites W2086172007 @default.
• W2088751510 cites W2087826959 @default.
• W2088751510 cites W2095145098 @default.
• W2088751510 cites W2098875742 @default.
• W2088751510 cites W2111515598 @default.
• W2088751510 cites W2131655970 @default.
• W2088751510 cites W2162166182 @default.
• W2088751510 doi "https://doi.org/10.1074/jbc.m208755200" @default.
• W2088751510 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12361946" @default.
• W2088751510 hasPublicationYear "2002" @default.
• W2088751510 type Work @default.
• W2088751510 sameAs 2088751510 @default.
• W2088751510 citedByCount "29" @default.
• W2088751510 countsByYear W20887515102012 @default.
• W2088751510 countsByYear W20887515102013 @default.
• W2088751510 countsByYear W20887515102014 @default.
• W2088751510 countsByYear W20887515102017 @default.
• W2088751510 countsByYear W20887515102018 @default.
• W2088751510 countsByYear W20887515102019 @default.
• W2088751510 countsByYear W20887515102022 @default.
• W2088751510 crossrefType "journal-article" @default.
• W2088751510 hasAuthorship W2088751510A5005931821 @default.
• W2088751510 hasAuthorship W2088751510A5017251809 @default.
• W2088751510 hasAuthorship W2088751510A5024941370 @default.
• W2088751510 hasBestOaLocation W20887515101 @default.
• W2088751510 hasConcept C11960822 @default.
• W2088751510 hasConcept C12554922 @default.
• W2088751510 hasConcept C132149769 @default.
• W2088751510 hasConcept C184235292 @default.
• W2088751510 hasConcept C185592680 @default.
• W2088751510 hasConcept C18903297 @default.
• W2088751510 hasConcept C2777289219 @default.
• W2088751510 hasConcept C55493867 @default.
• W2088751510 hasConcept C86803240 @default.
• W2088751510 hasConcept C87325107 @default.
• W2088751510 hasConcept C90934575 @default.
• W2088751510 hasConcept C95444343 @default.
• W2088751510 hasConcept C97029542 @default.
• W2088751510 hasConceptScore W2088751510C11960822 @default.
• W2088751510 hasConceptScore W2088751510C12554922 @default.
• W2088751510 hasConceptScore W2088751510C132149769 @default.
• W2088751510 hasConceptScore W2088751510C184235292 @default.
• W2088751510 hasConceptScore W2088751510C185592680 @default.
• W2088751510 hasConceptScore W2088751510C18903297 @default.
• W2088751510 hasConceptScore W2088751510C2777289219 @default.
• W2088751510 hasConceptScore W2088751510C55493867 @default.
• W2088751510 hasConceptScore W2088751510C86803240 @default.
• W2088751510 hasConceptScore W2088751510C87325107 @default.
• W2088751510 hasConceptScore W2088751510C90934575 @default.
• W2088751510 hasConceptScore W2088751510C95444343 @default.
• W2088751510 hasConceptScore W2088751510C97029542 @default.
• W2088751510 hasIssue "49" @default.
• W2088751510 hasLocation W20887515101 @default.
• W2088751510 hasOpenAccess W2088751510 @default.
• W2088751510 hasPrimaryLocation W20887515101 @default.
• W2088751510 hasRelatedWork W1557660156 @default.
• W2088751510 hasRelatedWork W175922642 @default.
• W2088751510 hasRelatedWork W1970474015 @default.
• W2088751510 hasRelatedWork W1973530967 @default.
• W2088751510 hasRelatedWork W2087084848 @default.
• W2088751510 hasRelatedWork W2352214813 @default.
• W2088751510 hasRelatedWork W2387788458 @default.
• W2088751510 hasRelatedWork W2491608603 @default.
• W2088751510 hasRelatedWork W2505862120 @default.
• W2088751510 hasRelatedWork W4223412920 @default.

• next