Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes

Gong, Huansheng; Murphy, Peter; Langille, Gavin; Minielly, Sarah J.; Murphy, Anne; McMaster, Christopher R.; Byers, David M.

doi:10.1016/j.bbapap.2008.07.017

Cited by 7 publications

(10 citation statements)

References 47 publications

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“…4B). We have recently shown that the L46W mutant of V. harveyi ACP undergoes a pronounced Mg 2ϩ -dependent fluorescence blue shift of emission maximum from ϳ355 nm to Ͻ320 nm, reflecting the movement of Trp-46 in helix II into the hydrophobic interior of the folded protein (15). This was also observed for the linL46W control protein in its apo form (Fig.…”

Section: Resultssupporting

confidence: 55%

“…The N-terminal splicing domain of the Ssp GyrB intein (residues 1-111, followed by a hepta-histidine tag; I N H) (31) was fused to the C terminus of ACP, while the C-terminal splicing domain (residues 393-435; I C ) was fused to the ACP N terminus (plasmid pTCYC-L46W). The L46W mutant of ACP was chosen because the tryptophan at position 46 (replacing leucine in the wild-type V. harveyi ACP) has been shown to be sensitive to the folding state of the protein, without affecting its secondary structure or substrate properties with a variety of ACP-dependent enzymes (15). After trans-splicing, the cyclic ACP L46W mutant (cycL46W) would therefore have its N and C termini connected by a three amino acid linker (Gly-Ser-Ala) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein Expression and Purification-To produce linL46W or linF50A protein, BL21(DE3)pLysS cells harboring the pGEX-linL46W (15) or pGEX-F50A (20) plasmid were grown to mid-log phase (A 595 ϭ 0.5-0.6) in LB medium (containing 100 g/ml ampicillin and 34 g/ml chloramphenicol) at 37°C and induced with 1 mM IPTG at 30°C to an A 660 of 1.5-2.0. Cells were harvested and protein purified as previously (20) with the additional cell lysis step using progressively smaller bore needles prior to sonication.…”

Section: Methodsmentioning

confidence: 99%

“…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 mobility in the long loop I connecting helices I and II, in the helix II-helix III region, and at the N and C termini, which are in close proximity.…”

Section: Bacterial Acyl Carrier Protein (Acp)mentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Bacterial Acyl Carrier Protein (Acp)mentioning

confidence: 99%

See 2 more Smart Citations

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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“…Intermediate to these two extremes, Helicobacter pylori ACP is partially folded under the same circumstances (16). As such, VhACP has been described as a natively unfolded protein, in particular because it has recently been shown that folding can be induced by binding to FAS enzymes (3,17). Various other factors have also been found to stabilize VhACP and result in protein folding.…”

mentioning

confidence: 99%

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H

Chan

Chu

Lau

et al. 2010

Journal of Biological Chemistry

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Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca 2؉ and Mg 2؉ and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ϳ20°C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by 15 N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.

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Determination of L-tyrosine Based on Luminescence Quenching of Mn-Doped ZnSe Quantum Dots in Enzyme Catalysis System

Yü

Huang

2010

J Fluoresc

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In this paper, we attempted to develop a novel application of Mn-doped ZnSe quantum dots (Mn: ZnSe d-dots) as probes to detect L-tyrosine (L-Tyr). The bio-conjugates of horseradish peroxidase (HRP)-conjugated Mn: ZnSe d-dots were used in the enzyme catalyzed reaction of L-Tyr with H(2)O(2.) Compared with traditional CdTe QDs, Mn: ZnSe d-dots have better biocompatibility and less negative impact on enzyme catalyzed system. In HRP-conjugated Mn: ZnSe-L-Tyr-H(2)O(2) system, electron transfer occurred between Mn: ZnSe d-dots and HRP. It resulted in the luminescence quenching of the Mn: ZnSe d-dots., which can be used to detect L-Tyr. The coupling of efficient quenching of Mn: ZnSe d-dots photoluminescence (PL) and the effective enzyme-catalysis can afford a simple and sensitive method for L-Tyr detection. The Mn: ZnSe d-dots-enzyme catalyzed system displays great potential in the development of enzyme-based biosensing systems for various analytes.

show abstract

Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes

Cited by 7 publications

References 47 publications

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

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

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H

Determination of L-tyrosine Based on Luminescence Quenching of Mn-Doped ZnSe Quantum Dots in Enzyme Catalysis System

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