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R J Fletterick

Publications and source records attributed to R J Fletterick.

At least 19 recordsLinked to original sources
PubMed1992

Purification of glycogen phosphorylase isozymes by metal-affinity chromatography.

Mammalian phosphorylase isozymes from muscle, brain and liver were expressed in Escherichia coli and purified from the crude bacterial cell extracts in one step using a copper-loaded, metal-affinity matrix. Good chromatographic behavior, enzyme activity and protein stability were maintained by judicious choice of pH and buffer which contained 250 mM sodium chloride and 25 mM beta-glycerophosphate at pH 7.0. Small amounts of beta-mercaptoethanol and EDTA in the buffers further stabilized the enzymes, but stripped some of the metal from the column which, nonetheless, retained good chromatographic characteristics. Owing to the presence of multiple surface histidine residues in the phosphorylase dimers, good enzyme purities (90-98%) and recoveries (>90%) were routinely obtained from crude bacterial lysates after two passes through the copper column. Of the various metal ions which were investigated, Cu2+ gave the best chromatographic results. Imidazole gradients at constant pH were used to selectively desorb the phosphorylase from the metal column whose capacity for phosphorylase binding in the presence of bacterial proteins exceeded 30 mg enzyme per milliliter of matrix.

Animals↗
PubMed1992

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1992

Tracking conformational states in allosteric transitions of phosphorylase.

An intrinsic molecular property of a protein domain can be determined by calculating its principal axes from the inertia tensor matrix. The mass-weighted principal axes can be used to calculate an ellipsoid representing the shape of the protein domain, providing an easy means of visualizing domain movements. Most importantly, the mass-weighted principal axes provide an intuitive means of characterizing domain relationships within a protein, as well as the disposition of domains in different protein conformers. Thus, this method provides a simple, quantitative description of differences of domain positions within various protein structures. We show the utility of this method by characterizing the quaternary and tertiary differences as observed in eight structures of phosphorylated or dephosphorylated glycogen phosphorylase with different effectors bound. This analysis revealed domain movements which were characteristic of the activated phosphorylase structures. The monomers of the phosphorylase dimer were found to move apart by a 2.5-A translation and to rotate apart, in three orthogonal directions, by a minimum of 3.2 degrees. Analysis of the three domains within the phosphorylase monomer showed that both simple and complex domain movements occur and that multiple domain configurations are energetically stable. We suggest that the C-terminal domain of phosphorylase moves along a simple path in the transition from an inactive to active conformation. The direction of translation and rotation is consistent, but the magnitude is variable. In contrast, this analysis showed that the activation domain did not behave as a rigid body, and therefore, the motion of this domain is not as easily characterized.

Allosteric Regulation↗
PubMed1992

The crystal structure of the Bacillus lentus alkaline protease, subtilisin BL, at 1.4 A resolution.

The crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 A resolution. The structure was solved by molecular replacement starting with the 2.1 A structure of subtilisin BPN' followed by molecular dynamics refinement using X-PLOR. A final crystallographic R-factor of 19% overall was obtained. The enzyme possesses stability at high pH, which is a result of the high pI of the protein. Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.). Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges. All of the potential partners are arginine with a pK around 12. Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes. Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI). The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence. The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are. Finally, one third of the serine side-chains in the protein have multiple conformations. This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure.

Amino Acid Sequence↗
PubMed1992

Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae.

Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from S. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.

Amino Acid Sequence↗
PubMed1992

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.

Amino Acid Sequence↗
PubMed1992

Engineered metalloregulation in enzymes.

Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.

Amino Acid Sequence↗
PubMed1992

Phosphorylase: a biological transducer.

A transducer is a device that receives energy from one system and transmits it, often in a different form, to another. Glycogen phosphorylase receives information from the cell or organism in the form of metabolic signals. The energy associated with the binding of these ligand signals is integrated and transmitted at an atomic level, allowing precise adjustment of the enzymatic activity. Understanding this elegant allosteric control has required several different approaches, but the structural requirements of allostery are being defined.

Allosteric Regulation↗
PubMed1991

Structural basis for the activation of glycogen phosphorylase b by adenosine monophosphate.

The three-dimensional structure of the activated state of glycogen phosphorylase (GP) as induced by adenosine monophosphate (AMP) has been determined from crystals of pyridoxalpyrophosphoryl-GP. The same quaternary changes relative to the inactive conformation as those induced by phosphorylation are induced by AMP, although the two regulatory signals function through different local structural mechanisms. Moreover, previous descriptions of the phosphorylase active state have been extended by demonstrating that, on activation, the amino- and carboxyl-terminal domains of GP rotate apart by 5 degrees, thereby increasing access of substrates to the catalytic site. The structure also reveals previously unobserved interactions with the nucleotide that accounts for the specificity of the nucleotide binding site for AMP in preference to inosine monophosphate.

Adenosine Monophosphate↗
PubMed1991

Expression of the protease inhibitor ecotin and its co-crystallization with trypsin.

We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.

Bacterial Proteins↗
PubMed1991

An engineered liver glycogen phosphorylase with AMP allosteric activation.

Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.

Adenosine Monophosphate↗
PubMed1991

Crystal structure of rat trypsin-S195C at -150 degrees C. Analysis of low activity of recombinant and semisynthetic thiol proteases.

The X-ray crystal structure of trypsin-S195C, a rat anionic trypsin mutant in which the active site serine has been replaced by cysteine, was determined at -150 degrees C and room temperature to 1.6 A resolution, R = 15.4% and 1.8 A resolution, R = 15.0%, respectively. Cryo-crystallography was employed to improve the quality of the diffraction data and the resulting structure by eliminating radiation damage and decreasing atomic thermal motion. The average temperature factor decreased by 10 A2 relative to that of the room temperature structure. No radiation-induced decay of the data was detected. The side-chains of the catalytic cysteine and histidine of trypsin-S195C are found with 25% occupancy in secondary orientations rotated 104 degrees and 90 degrees out of the active site, respectively. These alterations, as well as more subtle changes in the active site may be caused by the oxidation of the catalytic sulfur to sulfenic acid. The position of the carbonyl carbon of the tetrahedral intermediate analog, p-amidinophenylpyruvic acid, modeled into trypsin-S195C, is 1.1 A from the catalytic sulfur. The large size and altered approach of the catalytic sulfur to substrates could account for the observed low catalytic activity relative to wild-type trypsin. In addition to the benzamidine in the specificity pocket, two additional binding sites for benzamidine are characterized. One of these mediates an intermolecular contact that appears to maintain the crystal lattice.

Amino Acid Sequence↗
PubMed1991

Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis.

The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.

Amino Acid Sequence↗
PubMed1991

The sequence and reactive site of ecotin. A general inhibitor of pancreatic serine proteases from Escherichia coli.

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unusual in its ability to inhibit chymotrypsin, trypsin, and elastase. To address the structural basis of its broad specificity, the gene for ecotin has been cloned and its sequence determined. A promoter of the 17-base pair spacing class was identified, and the probable transcriptional start site lies 18 base pairs upstream from a ribosome binding locus. The gene is followed by a series of conserved repetitive extragenic palindromic sequences. Ecotin has a signal peptide of 20 amino acids which confirms its periplasmic localization. Sequence analyses by Edman degradation and mass spectrometry confirmed 71% of the deduced protein sequence of calculated monomeric molecular mass 16,096 Da. Comparisons of the primary structure for the 142-amino acid protein with the major classes of serine protease inhibitors suggest that ecotin is a novel inhibitor. The reactive site of ecotin was determined to be Met84 for its complexes with chymotrypsin, trypsin, and elastase. The scissile Met84-Met85 bond lies within a disulfide-bonded protein segment similar to other classes of inhibitors.

Amino Acid Sequence↗
PubMed1991

Hydrogen bonds involving sulfur atoms in proteins.

Intrachain hydrogen bonds are a hallmark of globular proteins. Traditionally, these involve oxygen and nitrogen atoms. The electronic structure of sulfur is compatible with hydrogen bond formation as well. We surveyed a set of 85 high-resolution protein structures in order to evaluate the prevalence and geometry of sulfur-containing hydrogen bonds. This information should be of interest to experimentalists and theoreticians interested in protein structure and protein engineering.

Animals↗
PubMed1991

Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli.

In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism. Essential to such an investigation is the ability to obtain large amounts of variant proteins. We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria. Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions. However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase. The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used. New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures. The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle. The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous. Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli. Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.

Animals↗
PubMed1990

Purification and crystallization of 15-lipoxygenase from rabbit reticulocytes.

We report a new purification of rabbit reticulocyte 15-lipoxygenase that has resulted in the first crystallization of a mammalian lipoxygenase. The enzyme was purified to homogeneity (greater than 98% pure by SDS-PAGE) using high pressure liquid chromatography on hydrophobic-interaction, hydroxyapatite and cation-exchange columns. Crystals were grown by the vapor diffusion method from concentrated solutions of the protein in sodium phosphate buffer, pH 7.0. The hexagonal, rod-shaped crystals were on average 0.09 mm x 0.09 mm x 0.4 mm, with approximate unit cell dimensions of a = b = 260 A, c = 145 A. The crystals diffract to 5 A resolution.

Animals↗