Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a ¹⁹F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific ¹⁹F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on ¹⁹F spins, a standard curve for ¹⁹F-tfmF chemical shifts was drawn for varying solvent H₂O/D₂O ratios. Further site-specific ¹⁹F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.     

Site-specific PEGylation of proteins containing unnatural amino acids

Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast. The azido group was used in a mild [3+2] cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein. This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.     

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.     

Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.     

Introduction of unnatural amino acids into proteins using expressed protein ligation

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.     

Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded 15N/19F-labeled unnatural amino acid

SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for ¹⁵N/¹⁹F-trifluoromethyl-phenylalanine (¹⁵N/¹⁹F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide (¹⁵N)¹H and side-chain ¹⁹F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide (¹⁵N)¹H and side-chain ¹⁹F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.     

Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered.     

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.     

Structural information on a membrane transport protein from nuclear magnetic resonance spectroscopy using sequence-selective nitroxide labeling.

The lactose transport protein (LacS) from Streptococcus thermophilus bearing a single cysteine mutation, K373C, within the putative interhelix loop 10-11 has been overexpressed in native membranes. Cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy (NMR) could selectively distinguish binding of (13)C-labeled substrate to just 50-60 nmol of LacS(K373C) in the native fluid membranes. Nitroxide electron spin-label at the K373C location was essentially immobile on the time scale of both conventional electron spin resonance spectroscopy (ESR) (<10(-8)s) and saturation-transfer ESR (<10(-3)s), under the same conditions as used in the NMR studies. The presence of the nitroxide spin-label effectively obscured the high-resolution NMR signal from bound substrate, even though (13)C-labeled substrate was shown to be within the binding center of the protein. The interhelix loop 10-11 is concluded to be in reasonably close proximity to the substrate binding site(s) of LacS (<15 A), and the loop region is expected to penetrate between the transmembrane segments of the protein that are involved in the translocation process.     

Sense codon-dependent introduction of unnatural amino acids into multiple sites of a protein

Cell-free protein synthesis, driven by a crude S30 extract from Escherichia coli, has been applied to the preparation of proteins containing unnatural amino acids at specific positions. We have developed methods for inactivating tRNA(Asp) and tRNA(Phe) within a crude E. coli tRNA by an antisense treatment and for digesting most of the tRNA within the S30 extract without essential damage to the ribosomal activity. In the present study, we applied these methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg(2+), the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low. The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. The results suggest that a good portion of the translation products are substituted at all of the seven positions originally occupied by Asp or Phe.     

19F nuclear magnetic resonance studies of selectively fluorinated derivatives of G- and F-actin

G-Actin is a globular protein (Mr 42 300) known to have three cysteine residues that are at least partially exposed and chemically reactive (Cys-10, -284, and -374). When G-actin was reacted with 3-bromo-1,1,1-trifluoropropanone, three resolvable 19F resonances were observed in the 19F NMR spectrum. This fluorinated G-actin derivative remained fully polymerizable, and its 31P NMR spectrum was not significantly different from that of unmodified G-actin, indicating that the chemical modification did not denature the actin and the modified residues do not interfere with the extent of polymerization or the binding of adenosine 5'-triphosphate. One of the three 19F resonances was assigned to fluorinated Cys-374 on the basis of its selective reaction with N-ethylmaleimide. This resonance was dramatically broadened after polymerization of fluorinated G-actin, while the other two resonances were not markedly broadened or shifted. Thus, Cys-10 and -284 are not involved in or appreciably affected by the polymerization of G-actin, while the mobility of the 19F label at Cys-374 is markedly reduced.     

19F nuclear magnetic resonance studies of free calcium in heart cells.

19F nuclear magnetic resonance is used in conjunction with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBapta), a fluorinated calcium chelator, to report steady-state intracellular free calcium levels ([Ca2+]i) in populations of resting, quiescent, isolated adult heart cells. 31P nuclear magnetic resonance shows that 5FBapta-loaded cells maintain normal intracellular high-energy phosphates, pH, and free Mg2+. The intracellular free calcium concentration of well perfused, isolated heart cells is 61 +/- 5 nM, measured with 5FBapta, which has a dissociation constant (Kd) for calcium chelation of 500 nM. A similar value is obtained with Quin-MF, another fluorinated calcium chelator with Kd and maximum calcium sensitivity at 80 nM. We find that the steady-state level of intracellular free calcium is increased by decreased extra-cellular sodium concentration, omission of extracellular magnesium, decreased extracellular pH, hyperglycemia, and upon treatment with lead acetate. Further, extracellular ATP caused a large transient increase in [Ca2+]i. Thus, while heart cells maintain a very low level of intracellular free Ca2+, acute alterations in extracellular environment can cause derangement of calcium homeostasis, resulting in measurable increases in [Ca2+]i.     

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.     

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca.

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.     

The use of unnatural amino acids to study and engineer protein function

1. Download : Download high-res image (194KB)
2. Download : Download full-size image