Aaron B Cowley

University of Kansas, Lawrence, KS, United States

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Publications (13)42.12 Total impact

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    ABSTRACT: In Pseudomonas aeruginosa, the algH gene regulates the cellular concentrations of a number of enzymes and the production of several virulence factors, and is suggested to serve a global regulatory function. The precise mechanism by which the algH gene product, the AlgH protein, functions is unknown. The same is true for AlgH family members from other bacteria. In order to lay the groundwork for understanding the physical underpinnings of AlgH function, we examined the structure and physical properties of AlgH in solution. Under reducing conditions, results of NMR, electrophoretic mobility, and sedimentation equilibrium experiments indicate AlgH is predominantly monomeric and monodisperse in solution. Under non-reducing conditions intra- and intermolecular disulfide bonds form, the latter promoting AlgH oligomerization. The high-resolution solution structure of AlgH reveals alpha/beta-sandwich architecture fashioned from ten beta strands and seven alpha helices. Comparison with available structures of orthologues indicates conservation of overall structural topology. The region of the protein most strongly conserved structurally also shows the highest amino acid sequence conservation and, as revealed by hydrogen-deuterium exchange studies, is also the most stable. In this region, evolutionary trace analysis identifies two clusters of amino acid residues with the highest evolutionary importance relative to all other AlgH residues. These frame a partially solvent exposed shallow hydrophobic cleft, perhaps identifying a site for intermolecular interactions. The results establish a physical foundation for understanding the structure and function of AlgH and AlgH family proteins and should be of general importance for further investigations of these and related proteins. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 04/2015; 83(6). DOI:10.1002/prot.24811 · 2.92 Impact Factor
  • Lijun Wang, Aaron B Cowley, David R Benson
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    ABSTRACT: Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.
    Protein Engineering Design and Selection 11/2007; 20(10):511-20. DOI:10.1093/protein/gzm053 · 2.32 Impact Factor
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    ABSTRACT: We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.
    Proteins Structure Function and Bioinformatics 05/2007; 67(2):293-304. DOI:10.1002/prot.21250 · 2.92 Impact Factor
  • Aaron B Cowley, David R Benson
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    ABSTRACT: We have recently reported that aquo and thioether complexes of the ferric cytochrome c heme peptide N-acetylmicroperoxidase-8 (FeIII-1) exhibit greater low-spin character than do the corresponding complexes of a synthetic, water-soluble, monohistidine-ligated heme peptide (FeIII-2; Cowley, A. B.; Lukat-Rodgers, G. S.; Rodgers, K. R.; Benson, D. R. Biochemistry 2004, 43, 1656-1666). Herein we report results of studies showing that weak-field ligands bearing a full (fluoride, chloride, hydroxide) or partial (phenoxide, thiocyanate) negative charge on the coordinating atom trigger dissociation of the axial His ligand in FeIII-2 but not in FeIII-1. We attribute the greater sensitivity of His ligation in FeIII-1 to weak-field anionic ligands than to weak-field neutral ligands to the following phenomena: (1) anionic ligands pull FeIII further from the mean plane of a porphyrin than do neutral ligands, which will have the effect of straining the His-Fe bond in FeIII-2, and (2) heme in FeIII-2 is likely to undergo a modest doming distortion following anion binding that will render the His-ligated side of the porphyrin concave, thereby increasing porphyrin/ligand steric interactions. We propose that ruffling of the heme in FeIII-1 is an important factor contributing to its ability to resist His dissociation by weak-field anions. First, ruffling should allow His to more closely approach the porphyrin than is possible in FeIII-2, thereby reducing bond strain following anion binding. Second, the ruffling deformation in FeIII-1, which is enforced by the double covalent heme-peptide linkage, will almost certainly prevent significant porphyrin doming.
    Inorganic Chemistry 02/2007; 46(1):48-59. DOI:10.1021/ic060682c · 4.79 Impact Factor
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    ABSTRACT: We describe detailed studies of peptide-sandwiched mesohemes PSMA and PSMW, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSMA with tryptophan (Trp) to give PSMW generates additional energy via Trp side chain-porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His-FeIII ligation to a greater extent than His-FeII ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (Em,8 value). This is intriguing because converting PSMA to PSMW decreased heme solvent exposure, which would normally be expected to stabilize FeII relative to FeIII. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b5. Our studies of the PSMs also revealed that reduction of FeIII to FeII reversed the relative affinities of the first and second His ligands for Fe (K2III > K1III; K2II < K1II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which FeII can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as "dynamic strain", is that an exogenous ligand can compete with one of the His ligands in an FeII-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b5. Exclusive binding of exogenous ligands by the FeII form of PSMA led to positive shifts in its Em,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.
    Inorganic Chemistry 12/2006; 45(25):9985-10001. DOI:10.1021/ic052205k · 4.79 Impact Factor
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    ABSTRACT: The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.
    Protein Engineering Design and Selection 01/2006; 18(12):571-9. DOI:10.1093/protein/gzi067 · 2.32 Impact Factor
  • Aaron B Cowley, Na Sun, Mario Rivera, David R Benson
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    ABSTRACT: The outer mitochondrial membrane isoform of mammalian cytochrome b(5) (OM b(5)) is distinguished from the microsomal isoform (Mc b(5)) by its considerably greater stability. In contrast, OM and Mc apocytochrome b(5) (apo-b(5)) exhibit similar thermodynamic stability. Contributing substantially to the greater stability of OM b(5) relative to that of Mc b(5) is the presence of Leu at position 71. Replacing Leu-71 in OM b(5) with the corresponding Mc b(5) residue (Ser) not only diminishes holoprotein stability but also markedly compromises apoprotein stability. The studies reported herein were undertaken to clarify the role played by Leu-71 in stabilizing OM b(5)s relative to Mc b(5)s, and were motivated by the possibility that stability is related to other differences in OM and Mc b(5) properties that are important for their specialized subcellular roles. The results of these studies show that Leu-71 plays an essential role in maintaining the structural integrity of the heme-independent folding core of OM apo-b(5) (core 2), despite its location in the disordered empty heme-binding pocket (core 1). The conformational integrity of core 2 in Mc apo-b(5)s is not similarly dependent on the presence of a hydrophobic residue at position 71, providing new evidence for evolution of compensating structural features not present in OM b(5)s. We propose that Leu-71 achieves its effect on OM apo-b(5) core 2 structure by participating in a nonspecific hydrophobic collapse of disordered core 1, templated by more conformationally restricted side chains of residues in the beta-sheet that separates the two cores. We hypothesize that this has the added effect of maintaining core 1 of OM apo-b(5)s in a state more compact than that which occurs in Mc apo-b(5)s, possibly contributing to stronger heme binding by limiting the number of non-native conformations that the empty heme-binding pocket can populate.
    Biochemistry 12/2005; 44(44):14606-15. DOI:10.1021/bi051337m · 3.19 Impact Factor
  • Journal of Biomolecular NMR 10/2005; 33(1):74. DOI:10.1007/s10858-005-1271-9 · 3.31 Impact Factor
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    ABSTRACT: The most common cause of mortality among cystic fibrosis sufferers is infection by antibiotic resistant strains of Pseudomonas aeruginosa. Means to control these strains continue to be an important goal. An integral component of the ability of many of these strains to defy antibiotic therapies is the protection afforded by the mucoexopolysaccharide alginate. Production of alginate by P. aeruginosa is tightly regulated at the transcriptional level. AlgH, a putative transcriptional regulator, is involved in regulating alginate biosynthesis as well as nucleoside diphosphate kinase activity and succinyl coenzyme A synthetase activity in P. aeruginosa. Sequence homologues are found in many bacterial species. Here, we describe a method for high level overexpression and high yield/high purity production of AlgH for biophysical and functional studies. The algH gene was cloned and AlgH was overexpressed in Escherichia coli using a commercially available vector with an inducible T7 promoter. We purified the recombinantly produced protein using a rapid classical purification scheme. The yield of purified protein, either isotopically labeled for NMR studies or unlabeled, is excellent (30-37 mg of purified protein per liter of minimal media culture), as is the purity (>95% pure). Analysis of the secondary structure using circular dichroism and NMR indicates that the protein is comprised of both beta-sheet and alpha-helical secondary structural elements. Heteronuclear NMR spectra indicate that AlgH is a monodisperse, folded globular protein. This rapid, high yield, and high purity method for AlgH production will permit further biophysical characterization of this protein including high resolution structural studies.
    Protein Expression and Purification 09/2005; 43(1):57-64. DOI:10.1016/j.pep.2005.02.017 · 1.51 Impact Factor
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    Aaron B Cowley, Mario Rivera, David R Benson
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    ABSTRACT: The microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b5 are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme-polypeptide interactions are stronger in OM b5s than in Mc b5s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structure-function relationships, because stronger heme-polypeptide interactions in OM b5s in comparison to Mc b5s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b5 persist when heme is removed, rendering the empty heme binding pocket of rOM apo-b5 more compact and less conformationally dynamic than that in bovine Mc (bMc) apo-b5. This may contribute to the stronger heme binding by OM apo-b5 by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo-b5 unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo-b5. We propose that a more robust beta-sheet in Mc apo-b5s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo-b5s.
    Protein Science 10/2004; 13(9):2316-29. DOI:10.1110/ps.04817704 · 2.86 Impact Factor
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    ABSTRACT: N-Acetylmicroperoxidase-8 (1) contains heme and residues 14-21 of horse mitochondrial cytochrome c (cyt c). The two thioether bonds linking protein to heme in cyt c are present in 1, and the native axial ligand His-18 remains coordinated to iron. As an approach to probing structural or functional roles played by the double covalent heme-protein linkage in cyt c, we have initiated a study in which the properties of 1 are compared with those of a synthetic mono-His coordinated heme peptide containing a single covalent linkage (2). One consequence of the greater conformational restriction imposed on peptide conformation in 1 is that His-Fe(III) coordination is approximately 1.4 kcal/mol more favorable in 1 than in 2. This highlights a clear advantage conferred to cyt c by having two covalent heme-protein linkages rather than one: greater thermodynamic stability of the protein fold. EPR (11 K) and resonance Raman (298 K) studies reveal that 1 and 2 exhibit a thermal high-spin/low-spin ferric equilibrium but that low-spin character is considerably more pronounced in 1. In addition, the thioether 2-(methylthio)ethanol (MTE) coordinates 0.5 kcal/mol more strongly to 1 than to 2 in 60:40 H(2)O/CH(3)OH and only triggers the expected conversion of iron to the low-spin state characteristic of ferric cyt c in the case of 1. This demonstrates that the axial ligand field provided by an imidazole and a thioether is too weak to induce a high-spin to low-spin conversion in a ferric porphyrin. Our results suggest that a conformationally constrained double covalent heme-protein linkage, as exists in 1 and its parent protein cyt c, is an effective solution that nature has evolved to circumvent this limitation. We propose that the stronger His-Fe(III) coordination enabled by such a linkage serves to markedly enhance the effective ligand field strength of His-18. Our studies with 1 and 2 suggest that a double covalent linkage in cyt c may also enable energetically more favorable trans ligation of Met-80 than would be possible if only a single linkage were present. This would serve to further increase the stability of the protein fold and perhaps to increase the effective ligand field strength of Met-80 as well.
    Biochemistry 03/2004; 43(6):1656-66. DOI:10.1021/bi035531p · 3.19 Impact Factor
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    ABSTRACT: Methionine-80 (Met-80) in mitochondrial cytochrome c (cyt c) can be oxidized to the corresponding sulfoxide by reactive oxygen species, a reaction of potential biological significance. As an approach to investigating how oxidation of Met-80 would influence its interactions with heme iron, we have examined binding of 2-(methylthio)ethanol (MTE) and dimethyl sulfoxide (DMSO), models for the side chains of Met and Met(SO), respectively, to ferrous and ferric N-acetylmicroperoxidase-8 (AcMP8). We find that DMSO coordinates 1.2 kcal/mol less strongly to Fe(III)-AcMP8 than does MTE, although both ligands form low-spin complexes. Comparison of spectroscopic data for the DMSO complex of Fe(III)-AcMP8 with published data for the Met(SO)-80 form of ferric cyt c allows us to conclude that Met(SO)-80 does not coordinate to iron in the latter. DMSO coordinates to Fe(II)-AcMP8 1.3 kcal/mol more strongly than does MTE, whereas Met-80 and Met(SO)-80 are reported to have approximately equal affinity for Fe(II) in cyt c. This result suggests that the steric environment near the heme iron in cyt c discriminates against coordination of Met(SO)-80. Vacuum quantum chemical density functional theory calculations confirm the greater affinity of the sulfoxide and show that coordination via oxygen is strongly favored. Resonance Raman spectroscopic data indicate that the preference for coordination via oxygen is maintained in solution. The computational data further indicate that the DMSO complex derives significant enthalpic stabilization from pi back-bonding but that iron to sulfur pi back-bonding does not make a significant contribution to bonding in the thioether complex.
    Inorganic Chemistry 12/2003; 42(23):7550-9. DOI:10.1021/ic034689v · 4.79 Impact Factor
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    ABSTRACT: As part of a larger effort to engineer the stability and hemin-binding properties of microsomal (Mc) cytochromes b(5) into rat liver outer mitochondrial membrane (OM) cytochrome (cyt) b(5), several mutants of rat OM cyt b(5) were prepared to study the effect of gradual and complete elimination of two extended hydrophobic networks, which are present in the structure of the mitochondrial protein and are absent in the structure of mammalian Mc cytochromes b(5). One of the hydrophobic networks, identified in a previous study [Altuve, A., Silchenko, S., Lee, K.-H., Kuczera, K., Terzyan, S., Zhang, X., Benson, D. R., and Rivera, M. (2001) Biochemistry 40, 9469-9483], encompasses the side chains of Ala-18, Ile-32, Leu-36, and Leu-47, whereas a second hydrophobic network, identified as part of this work, encompasses the side chains of Ile-25, Phe-58, Leu-71, and the heme. The X-ray structure of the A18S/I25L/I32L/L47R/L71S quintuple mutant of rat OM cyt b(5) demonstrates that both hydrophobic networks have been eliminated and that the corresponding structural elements of the Mc isoform have been introduced. The stability of the rat OM mutant proteins studied was found to decrease in the order wild type > I25L > A18S/I32L/L47R > L71S > A18S/I32L/L47R/L71S > 18S/I25L/I32L/L47R/L71S, indicating that the two hydrophobic networks do indeed contribute to the high stability of rat OM cyt b(5) relative to the bovine Mc isoform. Surprisingly, the quintuple mutant of rat OM cyt b(5) is less stable than bovine Mc cyt b(5), even though the former exhibits significantly slower rates of hemin release and hemin reorientation at pH 7.0. However, at pH 5.0 the bovine Mc and rat OM quintuple mutant proteins release hemin at comparable rates, suggesting that one or both of the His axial ligands in the rat OM protein are more resistant to protonation under physiological conditions. Results obtained from chemical denaturation experiments conducted with the apoproteins demonstrated that mutants containing L71S are significantly less stable than bovine Mc apocyt b(5), strongly suggesting that Leu-71 plays a pivotal role in the stabilization of rat OM apocyt b(5), presumably via hydrophobic interactions with Ile-25 and Phe-58. Because comparable interactions are absent in bovine Mc apocyt b(5), which contains Ser at position 71, it must resort to different interactions to stabilize its fold, thus highlighting yet another difference between rat OM and bovine Mc cyt b(5). During the course of these investigations we also discovered that rat OM cyt b(5) can be made to strongly favor hemin orientational isomer A (I32L) or isomer B (L71S) with a single point mutation and that release of hemin orientational isomers A and B can be kinetically resolved in certain rat OM mutants.
    Biochemistry 10/2002; 41(39):11566-81. DOI:10.1021/bi026005l · 3.19 Impact Factor

Publication Stats

132 Citations
42.12 Total Impact Points


  • 2004–2007
    • University of Kansas
      • Department of Chemistry
      Lawrence, KS, United States
  • 2003–2006
    • North Dakota State University
      • Department of Chemistry and Biochemistry
      Fargo, North Dakota, United States
  • 2005
    • University of Georgia
      • Department of Biochemistry and Molecular Biology
      Атина, Georgia, United States
  • 2002
    • Oklahoma State University - Stillwater
      • Department of Chemistry
      SWO, Oklahoma, United States