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Biomedical subjects

E E Scott

Publications and source records attributed to E E Scott.

11 recordsLinked to original sources
PubMed2001

A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function.

The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis.

Amino Acid Sequence↗
PubMed2001

The role of cytochrome 2B1 substrate recognition site residues 115, 294, 297, 298, and 362 in the oxidation of steroids and 7-alkoxycoumarins.

At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s. The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation. Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile. In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16alpha-hydroxytestosterone:16beta-hydroxytestosterone, with the most dramatic alteration being displayed by A298V. Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates. Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites. The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families.

Amino Acid Substitution↗
PubMed2000

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin.

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.

Amino Acid Substitution↗
PubMed2000

Mapping the pathways for O2 entry into and exit from myoglobin.

The effects of mutagenesis on geminate and bimolecular O2 rebinding to 90 mutants at 27 different positions were used to map pathways for ligand movement into and out of sperm whale myoglobin. By analogy to a baseball glove, the protein "catches" and then "holds" incoming ligand molecules long enough to allow bond formation with the iron atom. Opening of the glove occurs by outward movements of the distal histidine (His(64)), and the ligands are trapped in the interior "webbing" of the distal pocket, in the space surrounded by Ile(28), Leu(29), Leu(32), Val(68), and Ile(107). The size of this pocket is a major determinant of the rate of ligand entry into the protein. Immediately after photo- or thermal dissociation, O2 moves away from the iron into this interior pocket. The majority of the dissociated ligands return to the active site and either rebind to the iron atom or escape through the His(64) gate. A fraction of the ligands migrate further away from the heme group into cavities that have been defined as Xe binding sites 4 and 1; however, most of these ligands also return to the distal pocket, and net escape through the interior of wild-type myoglobin is <20-25%.

Animals↗
PubMed2000

The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis.

Apomyoglobins from 13 different mammals were examined for resistance to denaturation by guanidinium chloride. Unfolding was followed by circular dichroism and tryptophan fluorescence and analyzed globally using the two-step, three-state mechanism first described by Barrick and Baldwin (Barrick, D., and Baldwin, R. L. (1993) Biochemistry 32, 3790-3796). With one exception, the rise and fall of Trp fluorescence intensity correlates quantitatively with the native to intermediate to unfolded steps seen in the CD curves. Although the O(2) binding properties of the holoproteins are nearly identical, the unfolding transitions of the apomyoglobins show 600-fold differences in resistance to guanidinium chloride denaturation. Apomyoglobins from diving mammals, particularly from sperm whales, are the most stable, whereas the apoproteins from pig, horse, and sheep are the least stable, indicating selective pressure for resistance to denaturation in the whale proteins. Sequence comparisons suggest that the key stabilizing residues in whale globins are Ala(5), His(12), Ile(28), Thr(51), Ala(53), Ala(74), Lys(87), Lys(140), and Ile(142). Combinations of these residues were substituted into pig myoglobin. The resultant multiple mutants showed stabilities approaching that of recombinant sperm whale apomyoglobin. Thus, comparative mutagenesis can be used to increase heme protein stability and improve expression yields in bacteria without compromising function.

Amino Acid Sequence↗
PubMed1998

Stabilizing bound O2 in myoglobin by valine68 (E11) to asparagine substitution.

The isopropyl side chain of valine68 in myoglobin has been replaced by the acetamide side chain of asparagine in an attempt to engineer higher oxygen affinity. The asparagine replacement introduces a second hydrogen bond donor group into the distal heme pocket which could further stabilize bound oxygen. The Val68 to Asn substitution leads to approximately 3-fold increases in oxygen affinity and 4-6-fold decreases in CO affinity. As a result, the M-value (KCO/KO2) is lowered 15-20-fold to a value close to unity. An even larger enhancement of O2 affinity is seen when asparagine68 is inserted into H64L sperm whale myoglobin which lacks a distal histidine. The overall rate constants for oxygen and carbon monoxide binding to the single V68N myoglobin mutants are uniformly lower than those for the wild-type protein. In contrast, the overall rate constant for NO association is unchanged. Analyses of time courses monitoring the geminate recombination of ligands following nanosecond and picosecond flash photolysis of MbNO and MbO2 indicate that the barrier to ligand binding from within the heme pocket has been raised with little effect on the barrier to diffusion of the ligand into the pocket from the solvent. The crystal structures of the aquomet, deoxy, oxy, and carbon monoxy forms of the V68N mutant have been determined to resolutions ranging from 1.75 to 2.2 A at 150 K. The overall structures are very similar to those of the wild-type protein with the principal alterations taking place within and around the distal heme pocket. In all four structures the asparagine68 side chain lies almost parallel to the plane of the heme with its amide group directed toward the back of the distal heme pocket. The coordinated water molecule in the aquomet form and the bound oxygen in the oxy form can form hydrogen-bonding interactions with both the Asn68 amide group and the imidazole side chain of His64. Surprisingly, in the carbon monoxy form of the V68N mutant, the histidine64 side chain has swung completely out the distal pocket, its place being taken by two ordered water molecules. Overall, these functional and structural results show that the asparagine68 side chain (i) forms a strong hydrogen bond with bound oxygen through its -NH2 group but (ii) sterically hinders the approach of ligands to the iron from within the distal heme pocket.

Amino Acid Substitution↗
PubMed1998

Fused aminotetralins: novel antagonists with high selectivity for the dopamine D3 receptor.

Starting from a series of 2-aminotetralins 1, a novel series of N-[4-(4-phenylbenzoylamino)butyl]-octahydrobenzoquinolines and hexahydrobenzoindoles with high potency and selectivity for the dopamine D3 receptor has been designed. The effect of ligand chirality on binding affinity has been established. Selected derivatives (e.g. 2o, 2p) show high functional selectivity and enhanced in vivo properties compared to 1.

Animals↗
PubMed1997

Ligand migration in sperm whale myoglobin.

Geminate oxygen rebinding to myoglobin was followed from a few nanoseconds to a few microseconds after photolysis for more than 25 different oxymyoglobin point mutants in the presence and absence of 12 atm of xenon. In all cases, two relaxations were observed: an initial fast phase (half-time 20 ns) and a slower, smaller phase (half-time 0.5-2 micros). Generally, xenon accelerates the fast reaction but slows the slower reaction and diminishes its amplitude. The rates and proportions of the two components and the effects of xenon on them vary widely for different mutants. The locations of specific xenon binding sites [Tilton, R. F., Kuntz, I. D. Jr., and Petsko, G. A. (1984) Biochemistry 23, 2849-2857], the effects of point mutations on the geminate reactions, and molecular dynamics simulations were used to suggest locations in the protein interior occupied by ligands on the nanosecond to microsecond time scale. Photodissociated ligands may occupy xenon site 4 in the distal pocket and xenon site 1 below the plane of the heme. Rebinding from these positions corresponds to the slower geminate phase for O2 rebinding. The rapid geminate component is determined by competition between rebinding from a position closer to the iron atom and escape to solvent or more distant locations in the protein.

Animals↗
PubMed1993

Inhibition of polymorphonuclear leucocyte phagocytosis by Porphyromonas gingivalis culture products in patients with adult periodontitis.

Porphyromonas gingivalis culture products and a purified trypsin-like protease (TLPase) from the organism were tested for their effects on the phagocytosis of P. gingivalis by polymorphonuclear leucocytes (PMN) from 16 patients with adult periodontitis and 16 healthy subjects in a case-control study. Both the culture products (p < 0.0001) and the TLPase (p < 0.0001) significantly inhibited PMN phagocytosis by both case and control samples. Culture products were significantly more inhibitory in both cases (p < 0.0019) and controls (p < 0.0198) than that TLPase. The case PMNs were significantly more susceptible to inhibition by culture products than the control PMNs (p < 0.0238). The data suggest that patients with adult periodontitis have PMNs that are more susceptible than normal to the inhibitory effects of P. gingivalis and might be at greater risk than healthy subjects to infection by this pathogen.

Adult↗
PubMed1993

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50.

Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M(r) 115,000, M(r) 55,000 and M(r) 47,000 antigens together with a second M(r) 55,000 polypeptide which was a contaminant of the M(r) 55,000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M(r) 115,000 and M(r) 47,000 antigens being present in all strains tested. The distribution of the M(r) 55,000 antigens were slightly more restricted: one M(r) 55,000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M(r) 55,000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M(r) 115,000 and the outer membrane M(r) 55,000 antigen possess epitopes which are located on the surface of the bacterium.

Adult↗