Resolution of Tryptophan–ANS Fluorescence Energy Transfer in Apomyoglobin by Site-directed Mutagenesis¶

Sirangelo, Ivana; Malmo, Clorinda; Casillo, Mariateresa; Irace, Gaetano

doi:10.1562/0031-8655(2002)076<0381:rotafe>2.0.co;2

Cited by 15 publications

(14 citation statements)

References 31 publications

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“…Wild-type (WT) and mutant apoMbs were purified (data not shown) and correctly folded as suggested by their CD spectra under native conditions (Fig. Additionally, the mutations did not affect the function of apoMb as described previously [16,18,28]. Apparently, the mutations studied here did not affect the structure of the protein.…”

Section: Stability Of Wild-type and Mutantssupporting

confidence: 68%

“…Pre-fibrillar aggregates are usually more cytotoxic than mature fibrils [1,3] and apoMb pre-fibril species reduced the viability of N2A cells by about 50% (Fig. It is noteworthy that the W7F/W14F mutation causes amyloid formation in a His-tagged cloned apoMb even at physiological pH [28], but the mature amyloid fibril had an overall structure and cytotoxicity similar to that of WT. Moreover, cells incubated with either WT or mutant mature fibrils or pre-aggregates presented similar loss of viability (Fig.…”

Section: Cytotoxicitymentioning

confidence: 99%

“…(3C)). In the case of double mutant W7F/W14F, the two tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure, and their replacement decreases the stability of native apoMb by disrupting both the stability of helix A and its favorable packing with helix G [28]. In the double mutant H24V/H119F, two polar side-chains from the hydrophobic core were replaced by the hydrophobic side-chains of valine and phenylalanine, which have about the same vol-ume, strongly stabilizing the protein against acid-unfolding [18].…”

Section: Stability Of Wild-type and Mutantsmentioning

confidence: 99%

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Insights on the Structure of Amyloid Fibrils from Site-Directed Mutagenesis

Corrêa¹,

Ramos

2009

PPL

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To test the hypothesis that the ability to form ordered beta-rich amyloid fibers with identical structures is a generic property of proteins we present a study on the overall structures of fibers formed by apomyoglobin mutants that either stabilize or destabilize the native state or the intermediate. Our results indicate that, at least at the macroscopic level, ordered beta-rich amyloid fibers have similar structures.

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Section: Stability Of Wild-type and Mutantssupporting

confidence: 68%

Section: Cytotoxicitymentioning

confidence: 99%

Section: Stability Of Wild-type and Mutantsmentioning

confidence: 99%

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Insights on the Structure of Amyloid Fibrils from Site-Directed Mutagenesis

Corrêa¹,

Ramos

2009

PPL

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“…S2 in Supplementary Material) and (ii) a strong spectral overlap occurs between the emission of the Trp residue (donor) and the excitation of ANS (acceptor) [27], [36]. The binding of ANS was then assessed by measuring the decrease in the steady state emission intensity of Trp.…”

Section: Resultsmentioning

confidence: 99%

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

Henry

Wang

et al. 2012

PLoS ONE

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Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions.

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“…4A, inset). The observed quenching of intrinsic protein fluorescence by resonance energy transfer indicates that the donor (Trp) and the acceptor (ANS) are close in space (Sirangelo et al 2002). Because the Stern‐Volmer plot is nonlinear with an upward curvature (Fig.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution

Vamvaca¹,

Butz²,

Walter³

et al. 2005

Protein Science

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Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.Keywords: chorismate mutase; directed evolution; genetic selection; DNA shuffling; quaternary structure A large fraction of proteins in living cells exist as symmetric assemblies of multiple subunits (Goodsell and Olson 2000). Oligomerization provides advantageous properties often not available to the monomer, such as increased stability, new intersubunit binding sites, cooperativity, and allosteric regulation. To understand the chemical and genetic basis for the evolutionary transition between monomers and oligomers, several studies have focused on altering the quaternary structure of existing proteins by either reducing or increasing the number of their subunits (Mossing and Sauer 1990;Green et al. 1995;Dickason and Huston 1996; MacBeath et al. 1998b,c).In nature, infrequent but beneficial mutations are identified and propagated by genetic selection. This tool can also be exploited effectively in the laboratory to obtain novel protein oligomers starting from a diverse population of variants (Taylor et al. 2001a;Woycechowsky and Hilvert 2004). It is especially valuable in the case of enzymes, where structural changes are often poorly tolerated, and mutations that alter quaternary structure without causing a significant loss in catalytic activity are relatively rare.We have successfully used genetic selection to alter the topology of homodimeric AroQ chorismate mutases (CMs)-domain-swapped enzymes that catalyze a key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. For example, by inserting a random hinge-loop of eight residues into the H1 helix of the thermostable mutase from Methanococcus jannaschii (MjCM) and selecting for functional variants by genetic complementation, we obtained a monomeric catalyst with near native activity (MacBeath et al. 1998c). The topologically redesigned monomer unexpectedly exhibits the properties of a molten globule (Vamvaca et al. 2004), but structural ordering seen upon ligand binding indicates that protein folding and catalysis can be efficiently coupled.Reprint requests to: Donald Hilvert, Laboratorium fu¨r Organische Chemie, ETH Ho¨nggerberg/HCI F 339, CH-8093 Zu¨rich/Switzerland; e-mail: hilvert@org.chem.ethz...

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Resolution of Tryptophan–ANS Fluorescence Energy Transfer in Apomyoglobin by Site-directed Mutagenesis¶

Cited by 15 publications

References 31 publications

Insights on the Structure of Amyloid Fibrils from Site-Directed Mutagenesis

Insights on the Structure of Amyloid Fibrils from Site-Directed Mutagenesis

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution

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