Showing papers in "Biochemistry in 2003"

RNA interference in mammalian cells by chemically-modified RNA

Abstract: RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

The First Direct Characterization of a High-Valent Iron Intermediate in the Reaction of an α-Ketoglutarate-Dependent Dioxygenase: A High-Spin Fe(IV) Complex in Taurine/α-Ketoglutarate Dioxygenase (TauD) from Escherichia coli†

Abstract: The Fe(II)- and α-ketoglutarate(αKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O2 to a five-coordinate, (His)2(Asp)-facially coordinated Fe(II) center to which αKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O2 on the ketone carbonyl of αKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)O2-) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)O2- complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O2 has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/α-ke...

Metal Binding and Oxidation of Amyloid-β within Isolated Senile Plaque Cores: Raman Microscopic Evidence†

Abstract: Alzheimer's disease (AD) is characterized by the deposition of amyloid plaques in the parenchyma and vasculature of the brain. Although previous analytical studies have provided much information about the composition and structure of synthetic amyloid-beta fibrils, there is, surprisingly, a dearth of data on intact amyloid plaques from AD brain. Therefore, to elucidate the structure and detailed composition of isolated amyloid plaque cores, we utilized a high-resolution, nondestructive technique, Raman microscopy. The data are of very high quality and contain detailed information about protein composition and conformation, about post-translational modification, and about the chemistry of metal binding sites. Remarkably, spectra obtained for senile plaque (SP) cores isolated from AD brain are essentially identical both within and among brains. The Raman data show for the first time that the SP cores are composed largely of amyloid-beta and confirm inferences from X-ray studies that the structure is beta-sheet with the additional possibility that this may be present as a parallel beta-helix. Raman bands characteristic of methionine sulfoxide show that extensive methionine oxidation has occurred in the intact plaques. The Raman spectra also demonstrate that Zn(II) and Cu(II) are coordinated to histidine residues in the SP cores, at the side chains' N(tau) and N(pi) atoms, respectively. Treatment of the senile plaques with the chelator ethylenediaminetetraacetate reverses Cu binding to SP histidines and leads to a broadening of amide features, indicating a "loosening" of the beta-structure. Our results indicate that Abeta in vivo is a metalloprotein, and the loosening of the structure following chelation treatment suggests a possible means for the solubilization of amyloid deposits. The results also reveal a direct chemical basis for oxidative damage caused by amyloid-beta protein in AD.

Self-assembly of Aβ1-42 into globular neurotoxins

Abstract: Amyloid β 1−42 (Aβ1-42) is a self-associating peptide that becomes neurotoxic upon aggregation. Toxicity originally was attributed to the presence of large, readily formed Aβ fibrils, but a variety...

Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37.

Abstract: LL-37 is an amphipathic, α-helical, antimicrobial peptide. 15N chemical shift and 15N dipolar−shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with 31P NMR. The 31P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature s...

Zeroing in on the Pathogenic Form of α-Synuclein and Its Mechanism of Neurotoxicity in Parkinson's Disease†

Abstract: Parkinson's disease (PD) is linked to mutations in the protein α-synuclein, which can exist in vitro in several aggregation states, including a natively unfolded monomer, a β-sheet rich oligomer, or protofibril, and a stable amyloid fibril. This work reviews the current literature that is relevant to two linked questions: which of these species is pathogenic, and what is the mechanism of neurotoxicity? The amyloid fibril, fibrillar aggregates, Lewy bodies, and the α-synuclein monomer, which is normally expressed at high levels, are all unlikely to be pathogenic, for reasons discussed here. We therefore favor a toxic protofibril scenario, and propose that the pathogenic species is transiently populated during the process of fibrillization. Toxicity may arise from pore-like protofibrils that cause membrane permeabilization. An approach to testing this hypothesis is discussed.

Bipyrimidine Photoproducts Rather than Oxidative Lesions Are the Main Type of DNA Damage Involved in the Genotoxic Effect of Solar UVA Radiation

Abstract: Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer. UVA (320−340 nm) constitutes the large majority of solar UV radiation but is less effective than UVB (290−320 nm) at damaging DNA. Although UVA has been implicated in photocarcinogenesis, its contribution to sunlight mutagenesis has not been elucidated, and DNA damage produced by UVA remains poorly characterized. We employed HPLC−MS/MS and alkaline agarose gel electrophoresis in conjunction with the use of specific DNA repair proteins to determine the distribution of the various classes and types of DNA lesions, including bipyrimidine photoproducts, in Chinese hamster ovary cells exposed to pure UVA radiation, as well as UVB and simulated sunlight (λ > 295 nm) for comparison. At UVA doses compatible with human exposure, oxidative DNA lesions are not the major type of damage induced by UVA. Indeed, single-strand breaks, oxidized pyrimidines, oxidized purines (essentially 8-oxo-7,8-dihydroguanine), and cyclobutane pyrimidin...

The LOV domain family: photoresponsive signaling modules coupled to diverse output domains.

Abstract: For single-cell and multicellular systems to survive, they must accurately sense and respond to their cellular and extracellular environment. Light is a nearly ubiquitous environmental factor, and many species have evolved the capability to respond to this extracellular stimulus. Numerous photoreceptors underlie the activation of light-sensitive signal transduction cascades controlling these responses. Here, we review the properties of the light, oxygen, or voltage (LOV) family of blue-light photoreceptor domains, a subset of the Per-ARNT-Sim (PAS) superfamily. These flavin-binding domains, first identified in the higher-plant phototropins, are now shown to be present in plants, fungi, and bacteria. Notably, LOV domains are coupled to a wide array of other domains, including kinases, phosphodiesterases, F-box domains, STAS domains, and zinc fingers, which suggests that the absorption of blue light by LOV domains regulates the activity of these structurally and functionally diverse domains. LOV domains contain a conserved molecular volume extending from the flavin cofactor, which is the locus for light-driven structural change, to the molecular surface. We discuss the role of this conserved volume of structure in LOV-regulated processes.

Sequence analyses of G-protein-coupled receptors: similarities to rhodopsin.

Abstract: G-protein-coupled receptors (GPCRs) 1 constitute a large superfamily of receptor proteins responsible for signal transduction (see http://www.gpcr.org/7tm). Throughout all higher organisms, these receptors mediate recognition of environmental stimuli like light, odor, and taste, but also hormonal and other types of communications across plasma membranes (1). They are also important targets for pharmacological intervention via activating or blocking their action (2). Three families of GPCRs were identified, with family A being by far the largest (reviewed in refs 3-5). Its members are more closely related to each other within a few functional domains than those of the other families. In addition, numerous diseases have been linked to specific mutations within the genes encoding GPCRs, also making these receptors targets for specific therapeutic interventions including gene transfer (6-9).

Role of α-Synuclein Carboxy-Terminus on Fibril Formation in Vitro†

Abstract: Alpha-synuclein (α-syn) is the major component of intracellular inclusions in several neurodegenerative diseases, and the conversion of soluble α-syn into filamentous aggregates may contribute to d...

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite.

Abstract: Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.

2.1 A crystal structure of human PXR in complex with the St. John's wort compound hyperforin.

Abstract: The nuclear xenobiotic receptor PXR is activated by a wide variety of clinically used drugs and serves as a master regulator of drug metabolism and excretion gene expression in mammals. St. John's wort is used widely in Europe and the United States to treat depression. This unregulated herbal remedy leads to dangerous drug-drug interactions, however, in patients taking oral contraceptives, antivirals, or immunosuppressants. Such interactions are caused by the activation of the human PXR by hyperforin, the psychoactive agent in St. John's wort. In this study, we show that hyperforin induces the expression of numerous drug metabolism and excretion genes in primary human hepatocytes. We present the 2.1 A crystal structure of hyperforin in complex with the ligand binding domain of human PXR. Hyperforin induces conformational changes in PXR's ligand binding pocket relative to structures of human PXR elucidated previously and increases the size of the pocket by 250 A(3). We find that the mutation of individual aromatic residues within the ligand binding cavity changes PXR's response to particular ligands. Taken together, these results demonstrate that PXR employs structural flexibility to expand the chemical space it samples and that the mutation of specific residues within the ligand binding pocket of PXR tunes the receptor's response to ligands.

DNA interactions of monofunctional organometallic ruthenium(II) antitumor complexes in cell-free media.

Abstract: Modifications of natural DNA in a cell-free medium by antitumor monodentate Ru(II) arene compounds of the general formula [(η6-arene)Ru(en)Cl]+ (arene = biphenyl, dihydroanthracene, tetrahydroanthracene, p-cymene, or benzene; en = ethylenediamine) were studied by atomic absorption, melting behavior, transcription mapping, circular and linear dichroism, plasmid unwinding, competitive ethidium displacement, and differential pulse polarography. The results indicate that these complexes bind preferentially to guanine residues in double-helical DNA. The data are consistent with DNA binding of the complexes containing biphenyl, dihydroanthracene, or tetrahydroanthracene ligands that involves combined coordination to G N7 and noncovalent, hydrophobic interactions between the arene ligand and DNA, which may include arene intercalation and minor groove binding. In contrast, the single hydrocarbon rings in the p-cymene and benzene ruthenium complexes cannot interact with double-helical DNA by intercalation. Interes...

A Model of Structure and Catalysis for Ketoreductase Domains in Modular Polyketide Synthases

Abstract: A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr → Phe, Ser → Ala, and Lys → Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr → Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of ...

Nuclear Localization of α-Synuclein and Its Interaction with Histones†

Abstract: The aggregation of α-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders (“synucleinopathies”). However, the func...

A Lead-Dependent DNAzyme with a Two-Step Mechanism†

Abstract: A detailed biochemical and mechanistic study of in vitro selected variants of 8−17 DNAzymes is presented. Even though the 8−17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg2+ (called 8−17), 0.5 mM Mg2+/50 mM histidine (called Mg5), or 100 μM Zn2+ (called 17E), all variants are shown to be the most active with Pb2+ (8−17: kobs ∼0.5 min-1; Mg5: kobs ∼2 min-1; 17E: kobs ∼1 min-1 with 200 μM Pb2+ at pH 5.0). For the 17E variant of the 8−17 DNAzyme, the single-turnover rate constants followed the order of Pb2+ ≫ Zn2+ ≫ Mn2+ ≈ Co2+ > Ni2+ > Mg2+ ≈ Ca2+ > Sr2+ ≈ Ba2+. The catalytic rate is half-maximal at 13.5 μM Pb2+, 0.97 mM Zn2+, or 10.5 mM Mg2+, suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb2+ > Zn2+ > Mg2+. The Pb2+-dependent activity increases linearly with pH and the slope of the plot of log kobs versus pH is ∼1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variati...

Interfacial Anchor Properties of Tryptophan Residues in Transmembrane Peptides Can Dominate over Hydrophobic Matching Effects in Peptide−Lipid Interactions†

Abstract: Membrane model systems consisting of phosphatidylcholines and hydrophobic -helical peptides with tryptophan flanking residues, a characteristic motif for transmembrane protein segments, were used to investigate the contribution of tryptophans to peptide-lipid interactions. Peptides of different lengths and with the flanking tryptophans at different positions in the sequence were incorporated in relatively thick or thin lipid bilayers. The organization of the systems was assessed by NMR methods and by hydrogen/deuterium exchange in combination with mass spectrometry. Previously, it was found that relatively short peptides induce nonlamellar phases and that relatively long analogues order the lipid acyl chains in response to peptide-bilayer mismatch. Here it is shown that these effects do not correlate with the total hydrophobic peptide length, but instead with the length of the stretch between the flanking tryptophan residues. The tryptophan indole ring was consistently found to be positioned near the lipid carbonyl moieties, regardless of the peptide-lipid combination, as indicated by magic angle spinning NMR measurements. These observations suggest that the lipid adaptations are not primarily directed to avoid a peptide-lipid hydrophobic mismatch, but instead to prevent displacement of the tryptophan side chains from the polar-apolar interface. In contrast, long lysine-flanked analogues fully associate with a bilayer without significant lipid adaptations, and hydrogen/deuterium exchange experiments indicate that this is achieved by simply exposing more (hydrophobic) residues to the lipid headgroup region. The results highlight the specific properties that are imposed on transmembrane protein segments by flanking tryptophan residues.

Enzyme electrokinetics: using protein film voltammetry to investigate redox enzymes and their mechanisms.

Abstract: Protein film voltammetry is a relatively new approach to studying redox enzymes, the concept being that a sample of a redox protein is configured as a film on an electrode and probed by a variety of electrochemical techniques. The enzyme molecules are bound at the electrode surface in such a way that there is fast electron transfer and complete retention of the chemistry of the active site that is observed in more conventional experiments. Modulations of the electrode potential or catalytic turnover result in the movement of electrons to, from, and within the enzyme; this is detected as a current that varies in characteristic ways with time and potential. Henceforth, the potential dimension is introduced into enzyme kinetics. The presence of additional intrinsic redox centers for providing fast intramolecular electron transfer between a buried active site and the protein surface is an important factor. Centers which carry out cooperative two-electron transfer, most obviously flavins, produce a particularl...

The plant dehydrins: structure and putative functions.

Abstract: This review deals with recent data on the structure and biochemical properties of dehydrins, proteins that are normally synthesized in maturating seeds during their desiccation, and also in vegetative tissues of plants treated with abscisic acid or exposed to environmental stress factors that result in cellular dehydration The dehydrins are considered as stress proteins involved in formation of plant protective reactions against dehydration The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y-, S-, and K-segments The K-segment representing a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) forming amphiphilic α-helix has been found in all dehydrins The pathways of regulation of dehydrin gene expression, putative functions of dehydrins, and molecular mechanisms of their actions are discussed

C5L2, a Nonsignaling C5A Binding Protein†

Abstract: C5a anaphylatoxin, a potent inflammatory mediator, is known to act through a specific G protein coupled receptor. However, some of the complex effects of C5a in vivo may not be explained solely by the deletion of the known receptor. Here, we show that an orphan receptor, identified as C5L2, is a high affinity C5a binding protein. Unlike the previously described C5aR, C5L2 is obligately uncoupled from heterotrimeric G proteins, in part by virtue of an amino acid alteration in the so-called DRY sequence at the end of the third transmembrane segment. Both human and murine C5L2 bear a leucine for arginine replacement at this site. C5L2, when transfected into several cell types, is weakly phosphorylated in transfected cells following binding of C5a but does not induce significant activation of MAP kinases, mediate calcium flux, or stimulate chemotaxis. Bone marrow cells from wild type respond robustly to C5a with induction and suppression of a number of inflammation related genes. In contrast, C5a receptor deficient mice, which bear C5L2 alone, do not respond to C5a with changes in gene transcription by microarray analyses. Biophysical properties of the C5L2, including slow ligand on and off rates, absence of internalization, and relatively high affinity for the C5a des Arg metabolite, suggest that this receptor may serve to modulate C5a biological functions in vivo. Finally, in contrast to previous reports, we find absolutely no interaction of C5L2 with other anaphylatoxins C3a and C4a.

Copper coordination in the full-length, recombinant prion protein.

Abstract: The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991–4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760–13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92–96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin–echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.

The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.

Abstract: Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an Rfree of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A2 (cPLA2) [Dessen, A., et al. (1999) Cell 97, 349−360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA2 and unlike the canonical α/β-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initial...

Conformational Behavior and Aggregation of α-Synuclein in Organic Solvents: Modeling the Effects of Membranes†

Abstract: Intracellular proteinaceous inclusions (Lewy bodies and Lewy neurites) of α-synuclein are pathological hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple systemic atrophy. The molecular mechanisms underlying the aggregation of α-synuclein into such filamentous inclusions remain unknown, although many factors have been implicated, including interactions with lipid membranes. To model the effects of membrane fields on α-synuclein, we analyzed the structural and fibrillation properties of this protein in mixtures of water with simple and fluorinated alcohols. All solvents that were studied induced folding of α-synuclein, with the common first stage being formation of a partially folded intermediate with an enhanced propensity to fibrillate. Protein fibrillation was completely inhibited due to formation of β-structure-enriched oligomers with high concentrations of methanol, ethanol, and propanol and moderate concentrations of trifluoroethanol (TF...

Mechanism of Formin-Induced Nucleation of Actin Filaments

Abstract: A fragment of the yeast formin Bni1 containing the FH1FH2 domains increases the rate of filament nucleation from pure G-actin [Pruyne et al. (2002) Science 297, 612−615]. To determine the mechanism of nucleation, we compared the G-actin dependence of Bni1FH1FH2-induced polymerization with theoretical models. The data best fit a model suggesting that Bni1FH1FH2 stabilizes an actin dimer. We also show that nucleation increases with the square root of the Bni1FH1FH2 concentration. We demonstrate that this relationship is expected for any such nucleator, independent of nucleus size. The proline-rich FH1 domain binds profilin, and deletion of this domain decreases the contribution of profilin−actin to the nucleation. A role for profilin binding to the FH1 domain in filament nucleation was supported by the inability of Bni1FH1FH2 to utilize a mutant profilin, H133S profilin, with defective binding to polyproline. Bni1FH1FH2 partially inhibits barbed-end elongation, and we find that the rate constants for both p...

A new strategy for the site-specific modification of proteins in vivo.

Abstract: We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Native State Proline Isomerization: An Intrinsic Molecular Switch†

Abstract: Exquisite control of biological function is achieved via tight regulation of the catalytic and binding activities of cellular proteins. The mechanistic details of protein regulation vary from targeted chemical modification of amino acid side chains (1) to the quite drastic global unfolding of an entire polypeptide chain (2). Peptidyl prolyl cis/trans isomerization is emerging as a potentially general mechanism for the control of protein function (3). While most structures of native, folded proteins reveal peptidyl-prolyl imide bonds that adopt either the cis or trans conformation, there are a growing number of folded proteins that exhibit conformational heterogeneity about one or more peptidyl-prolyl bonds. Unlike covalent modification or global unfolding, proline isomerization is an intrinsic conformational exchange process that has the potential to direct ligand recognition and to control protein activity within the confines of the native state. Disciplines Biochemistry, Biophysics, and Structural Biology | Molecular Biology Comments Reprinted (adapted) with permission from Biochemistry 42 (2003), 9515, doi:10.1021/bi0350710. Copyright 2003 American Chemical Society. Rights One-time permission is granted only for the use specified in your request. No additional uses are granted (such as derivative works or other editions). For any other uses, please submit a new request. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/bbmb_ag_pubs/14

Vibrational Stark Effects Calibrate the Sensitivity of Vibrational Probes for Electric Fields in Proteins

Abstract: Infrared spectroscopy is widely used to probe local environments and dynamics in proteins. The introduction of a unique vibration at a specific site of a protein or more complex assembly offers many advantages over observing the spectra of an unmodified protein. We have previously shown that infrared frequency shifts in proteins can arise from differences in the local electric field at the probe vibration. Thus, vibrational frequencies can be used to map electric fields in proteins at many sites or to measure the change in electric field due to a perturbation. The Stark tuning rate gives the sensitivity of a vibrational frequency to an electric field, and for it to be useful, the Stark tuning rate should be as large as possible. Vibrational Stark effect spectroscopy provides a direct measurement of the Stark tuning rate and allows a quantitative interpretation of frequency shifts. We present vibrational Stark spectra of several bond types, extending our work on nitriles and carbonyls and characterizing four additional bond types (carbon-fluorine, carbon-deuterium, azide, and nitro bonds) that are potential probes for electric fields in proteins. The measured Stark tuning rates, peak positions, and extinction coefficients provide the primary information needed to design amino acid analogues or labels to act as probes of local environments in proteins.

Partially folded intermediates in insulin fibrillation.

Abstract: Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn·HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn·HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn·HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn·HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn·HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentration...