Refinement of the NMR structures for acyl carrier protein with scalar coupling data

Kim, Yangmee; Prestegard, James H.

doi:10.1002/prot.340080411

Cited by 168 publications

(124 citation statements)

References 36 publications

(3 reference statements)

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“…It is unclear whether this inhibition reflects interactions between the apoACP protein and MAT or KS/CLF or both. Since the solution structures of several Type II ACPs have been solved (31)(32)(33)(34)(35)(36)(37)(38), further studies into the structural basis for these molecular recognition properties could provide fundamentally new insights into the importance of protein-protein interactions in regulating Type II PKS function and specificity.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Dreier¹,

Shah²,

Khosla³

1999

Journal of Biological Chemistry

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Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA: ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-␤-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of proteinprotein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 ؎ 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [ 14 C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

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Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Dreier¹,

Shah²,

Khosla³

1999

Journal of Biological Chemistry

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“…Finally, the upper-bound limit for each distance constraint was increased 10%. Backbone -angle restraints, derived from 3 J NH-CH coupling constants, were determined by using the method of Kim and Prestegard (43). Restraints were restricted to Ϫ60 Ϯ 40 for coupling constants Ͻ7 Hz and to Ϫ120 Ϯ 50 for coupling constants Ͼ8 Hz.…”

mentioning

confidence: 99%

De novodesigned cyclic-peptide heme complexes

Rosenblatt

Wang

Suslick

2003

Proc. Natl. Acad. Sci. U.S.A.

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The structural characterization of de novo designed metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, we have prepared a series of cyclic peptides that bind to water-soluble metalloporphyrins (Fe III and Co III ). Neutral and positively charged histidine-containing peptides bind with a high affinity, whereas anionic peptides bind only weakly to the negatively charged metalloporphyrin. Additionally, it was found that the peptide becomes helical only in the presence of the metalloporphyrin. CD experiments confirm that the metalloporphyrin binds specific cyclic peptides with high affinity and with isodichroic behavior. Thermal unfolding experiments show that the complex has ''native-like'' properties. Finally, NMR spectroscopy produced well dispersed spectra and experimental restraints that provide a high-resolution solution structure of the complexed peptide.O ne approach to understanding the folding and function of proteins is to attempt their design from first principles (1-3). Such design, however, requires not only incorporation of nonpolar interactions, but also inclusion of specific hydrogen bonds, disulfide bridges, ion pairs, and metal chelation. Without these interactions, proteins form compact folding intermediates referred to as molten globules (4). Molten-globule structures are compact and possess a high degree of secondary structure, but they are also highly dynamic, with both internal and external side chains rapidly interconverting among a large family of rotamers. Analysis of protein structures, on the other hand, reveals that the interior hydrophobic core is well packed and rigid. Therefore, two challenges occur in the design of ''native-like'' proteins. By using positive design (3, 5-7), one can take into account issues such as hydrophobicity and the helix-or sheet-forming propensity of amino acids. By using negative design [a term developed to describe the selective introduction of unique stabilizing interactions (2, 3)], one can introduce features that stabilize one specific fold, while, at the same time, they destabilize alternative topologies. By using this dual strategy, one can increase the free-energy gap between the native and molten-globule states.With the challenge of design making rapid progress, several groups have begun to look at the incorporation of functional cofactors in hopes of producing de novo catalytic proteins (8-14). Many enzymes require mono-and dinuclear metal ions (15), metal clusters (ferredoxin and nitrogenase) (16), heme (cytochromes and globins) (17), and organic cofactors (flavin-and quinone-based enzymes) (18). These cofactors are important, especially when processes such as oxidation͞reduction or substrate binding and activation are required (15).Studies of natural enzymes have led to considerable understanding of their structure and function. Reduction of these complicated systems to minimal models, however, will allow ...

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“…Over two dozen nuclear magnetic resonance (NMR) and x-ray crystal structures have revealed a conserved "ACP fold" consisting of a four-helix bundle (1). Fatty acids covalently attached to the phosphopantetheine prosthetic group at the N-terminal end of helix II are enclosed within the hydrophobic interior of this bundle, interacting predominantly with residues on helices II-IV (2)(3)(4)(5). Further computational (6,7), crystallographic (8,9), and mutagenic (10 -13) analyses have implicated the acidic central helix II as a "recognition helix" for interaction with most of the ACP enzyme partners.…”

Section: Bacterial Acyl Carrier Protein (Acp)mentioning

confidence: 99%

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

Volkmann

Murphy

Rowland

et al. 2010

Journal of Biological Chemistry

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Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein. Bacterial acyl carrier protein (ACP)2 is a small (typically 70 -80 residue) protein required for the synthesis and transfer of fatty acyl chains in the production of phospholipids and other specialized products, including lipid A, lipoic acid, acylhomoserine lactones, and hemolysin (reviewed in Ref. 1). Over two dozen nuclear magnetic resonance (NMR) and x-ray crystal structures have revealed a conserved "ACP fold" consisting of a four-helix bundle (1). Fatty acids covalently attached to the phosphopantetheine prosthetic group at the N-terminal end of helix II are enclosed within the hydrophobic interior of this bundle, interacting predominantly with residues on helices II-IV (2-5). Further computational (6, 7), crystallographic (8, 9), and mutagenic (10 -13) analyses have implicated the acidic central helix II as a "recognition helix" for interaction with most of the ACP enzyme partners. Subtle conformational alterations in this region likely also contribute to discrimination by ACPdependent enzymes among the various acyl-ACP derivatives (4,14).The acidic nature of ACP (pI ϳ 4) contributes to a highly dynamic and flexible structure in solution, and some ACPs exhibit features characteristic of natively unfolded proteins (1). For example, Vibrio harveyi ACP is largely unfolded at neutral pH, but its helical conformation can be stabilized by charge neutralization (i.e. at low pH or by residue replacements) (13), by binding of divalent cations to helix II (10), or by interaction with partner enzymes (15). Molecular dynamic simulations (16) and NMR experiments (17, 18) have indicated greatest mobili...

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Refinement of the NMR structures for acyl carrier protein with scalar coupling data

Cited by 168 publications

References 36 publications

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

De novodesigned cyclic-peptide heme complexes

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

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