F NMR to Probe Biological Interactions of Proteins and Peptides

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Using 19F NMR to Probe Biological Interactions of Proteins and Peptides E. Neil G. Marsh†,‡,* and Yuta Suzuki†,§ †

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States

‡

ABSTRACT: Fluorine is a valuable probe for investigating the interactions of biological molecules because of its favorable NMR characteristics, its small size, and its near total absence from biology. Advances in biosynthetic methods allow fluorine to be introduced into peptides and proteins with high precision, and the increasing sensitivity of NMR spectrometers has facilitated the use of 19F NMR to obtain molecular-level insights into a wide range of often-complex biological interactions. Here, we summarize the advantages of solutionstate 19F NMR for studying the interactions of peptides and proteins with other biological molecules, review methods for the production of fluorine-labeled materials, and describe some representative recent examples in which 19F NMR has been used to study conformational changes in peptides and proteins and their interactions with other biological molecules.

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been used to investigate. For reasons of space, we have limited our discussion to solution-phase NMR; some excellent reviews on the use of solid state 19F NMR to study biochemical interactions, particularly in relation to membrane proteins, have been published.11,12 Lastly, we note that this is not by any means a comprehensive review of the literature, and we apologize to those colleagues whose work we may have overlooked due to space limitations. Advantages of 19F NMR for Studying Biological Molecules. The 19F nucleus is spin 1/2 and exists in 100% natural abundance; it has excellent NMR properties that are comparable to that of proton NMR. The large magnetogyric ratio translates into high sensitivity in 1D NMR spectroscopy (83% sensitivity relative to 1H). Strong dipolar coupling allows 19 F−19F and 1H−19F nuclear Overhauser effects to be measured.13,14 This technique has been less commonly applied to the study of proteins because line-broadening weakens the signals; however, as an example, homonuclear 19F−19F and heteronuclear 19F−1H NOEs between a fluorinated ligand and 4-trifluoromethylphenylglycine (4-tfmpGly)-labeled Bcl-xL, an antiapoptotic protein, were used to characterize the binding interaction.15 One of the most useful and simple features of the 19 F nucleus is the intrinsic sensitivity of its chemical shift to changes in the local chemical environment. 19F chemical shifts span ∼400 ppm whereas 1H chemical shifts span ∼15 ppm.16 Therefore, a wide range of changes affecting the local environment of a peptide or protein can be detected using

luorine has proved a remarkably useful element with which to study biological systems1−3a utility that derives in large part from its almost total absence from biology! Fluorine is often considered as isosteric with hydrogen because the van der Waals radius of fluorine is 1.35 Å, similar to that of hydrogen, 1.2 Å, although a C−F bond is actually significantly longer (∼1.4 Å) than a C−H bond (∼1.0 Å). Nevertheless, fluorine can often replace hydrogen in small molecules with minimal effect on their binding to enzymes and proteins.4 For this reason, fluorination has been utilized extensively by the pharmaceutical industry to improve the pharmacokinetics and bioavailability of drugs.5−7 On the other hand, highly fluorinated organic molecules possess physiochemical properties that are significantly different from their hydrocarbon counterparts (e.g., polyethylene and polytetrafluoroethylene). For this reason, there have been numerous studies that have explored the effects of incorporating highly fluorinated amino acids on the physical and biological properties of proteins and peptides with the objective of introducing novel characteristics into biological systems. 8−10 In general, however, the introduction of a limited number of fluorine atoms into a protein does not appear to greatly change its structure or biological activity. In this review, we focus on the use of fluorinated peptides and proteins to probe their interactions with other biological molecules, in studies that primarily exploit the favorable NMR properties of the 19F nucleus. First, we discuss some of the properties of the 19F nucleus that make it advantageous for studying biological macromolecules. Next, we briefly summarize methods available to prepare fluorine-labeled proteins and peptides. We then discuss some representative recent examples that illustrate a variety of biological problems that 19F NMR has © XXXX American Chemical Society

Received: February 14, 2014 Accepted: April 24, 2014

A

dx.doi.org/10.1021/cb500111u | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

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simple 1D NMR techniques by incorporating suitably labeled residues at appropriate positions within the protein. Protein conformational changes, ligand binding, and interactions with other proteins, nucleic acids or lipid membranes are all amenable to detection by 19F NMR chemical shift changes.3,17 The 19F chemical shift is also sensitive to solvent (H2O/D2O) isotope effects, which have been used to study structural changes in large proteins as well as peptide-membrane interactions.3 Information on protein dynamics can be obtained from measuring the relaxation properties (T1 and T2) of the 19F nucleus. The fact that fluorine is essentially absent from biological molecules means that there is no overlap from background signals, a problem that often afflicts NMR measurements using 1 H, 13C, and 15N nuclei. This advantage potentially allows 19F NMR spectroscopy to be used to study large multiprotein complexes as well as proteins in vivo, where signals from the protein of interest are often attenuated in other NMR techniques. Therefore, the incorporation of fluorine into biomolecules is an attractive tool to investigate protein conformational changes and dynamics, enzyme mechanism, and protein−ligand and protein−membrane interactions in biological systems. Although, when incorporated into macromolecules, line broadening of 19F peaks occur (which is also a problem with other nuclei) the large chemical shift dispersion reduces the problem of peak overlap. One obvious disadvantage of 19F NMR is the need to introduce fluorine into the protein of interest! Therefore, we briefly review some of the commonly used methods to accomplish this. Synthesis of Fluorinated Proteins and Peptides. Various fluorinated amino acids have been used as NMR probes, many of which are commercially available. The chemical structures of some of the 19F labeled amino acid analogues that have been utilized in 19F NMR studies in chemical biology are shown in Table 1. In many cases “lightly fluorinated” amino acids can be incorporated biosynthetically, as they are not discriminated by the cognate actyl-tRNA synthetases, a feature that facilitated pioneering studies of proteins by NMR.18,19 Examples include 3-fluorophenylalanine (3-fPhe),20,21 4-fluorophenylalanine (4-fPhe),22−24 5-fluorotryptophan (5-fTrp),25−29 6-fluorotryptophan (6-fTrp),23,30 and 3-fluorotyrosine (3-fTyr).18,19,31−38 Fluorinated aromatic amino acids, phenylalanine (Phe) and tryptophan (Trp) residues in particular, have been used to study conformational changes associated with protein folding. For example, 3-fTyr has also been utilized to monitor structural changes and examine surface exposure using solvent isotope-induced chemical shift changes between H2O and D2O.35 Fluorinated aliphatic amino acids including 5-fluoroleucine (5-fLeu),39 trifluoroethylglycine (tfeGly), 40,41 trifluoromethionine (tfMet),42−44 difluoromethionine,45 and 2-fluorohistidine (2fHis)46 have also been used as 19F reporters to examine protein conformational changes. Methods for the introduction of unnatural amino acids into proteins have been reviewed extensively47−50 and are summarized only briefly here. Whereas lightly fluorinated amino acids may be incorporated into proteins by including the fluorinated amino acid in the growth medium and using a suitable auxotrophic bacterium, the introduction of more highly fluorinated amino acids is less straightforward. The use of tRNA synthetases that have been mutated to relax their specificity toward their cognate amino acids has allowed a few extensively fluorinated amino acids such as hexafluor-

Table 1. Chemical Structures of Representative Fluorinated Amino Acids Utilized in 19F NMR Studies of Biological Systems

a Commercially available fluorinated amino acids. bAmino acid also available as other fluorinated analogues. cPostmodification of cysteine residues on protein. d3-fPhe was used in this study. References in bold refer to studies discussed in more detail the text.

oleucine to be incorporated biosynthetically, although this strategy is not site-specific.51 Specific incorporation of nonnatural amino acids can be achieved using an evolved orthogonal aminoacyl-tRNA synthase/tRNA pair.49 This technique has successfully been used to introduce fluorinated aromatic residues such as trifluoromethylphenylalanine (tfmPhe) at specific positions in large proteins.36,52−54 For small proteins and peptides, solid phase chemical synthesis is the method of choice because it offers the greatest flexibility with respect to both the identity and position of the noncanonical amino acid. When combined with native chemical ligation techniques,55−57 large proteins can potentially be synthesized that contain uniquely labeled residues. This strategy has recently been applied to synthesize affibody proteins containing perfluorinated phenyl rings.58 Lastly, B



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Figure 1. Conformation changes of β2AR to ligand binding determined by 19F NMR (a) The structure of active-state of β2AR in the complex with the agonist BI-167107 (PDB 3SN6). (b) Chemical structures, names and pharmacological efficacy of the ligands (left) and the relative population of active (red) and inactive (blue) states of β2AR measured by the 1D 19F NMR spectra (right). (c) Plot of the relative peak volumes for C265A versus C327A. The relative peak volume is calculated as the ratios of the active state volume and the sum of the active and inactive state volumes. Ligands are indicated as agonists (black circles with yellow background), biased ligands (red triangle with green background), and a neutral antagonist (black square), an inverse agonist (black diamond), and apo (open square). Images were reproduced and modified from ref 59 with permission; copyright 2012, American Academy for the Advancement of Science.

fluorinated probes can be introduced by chemical modification using fluorinated “tags” such as 2,2,2-trifluoroethanethiol (TFET) that are reactive toward nucleophilic side chains, most usually cysteinyl residues.3,59,60 These may be either naturally present in the wild-type protein or introduced by mutagenesis at specific locations within the structure. Although potentially more disruptive to the protein’s structure, this method provides the most-straightforward method of introducing highly fluorinated probes into large proteins. Applications of 19F NMR to Study Biological Phenomena. Protein−Small Molecule Interactions. The high sensitivity of the 19F nucleus to the surrounding environment provides a powerful tool to investigate the structural changes that accompany a protein binding to small molecules such as natural ligands or inhibitors. In an elegant study, conformational changes in the β2-adrenergic receptor (β2AR), in response to various agonists and antagonists binding were examined using 19F NMR.59 β2AR is a G protein coupled receptor that transmits signals from a wide range of ligands to its cytoplasmic partners (Figure 1). A small, nonperturbing 19F probe was introduced by the covalent modification of three native Cys residues in the cytoplasmic regions of the receptor using TFET. This allowed subtle conformational changes in the cytoplasmic ends of two α-helices in response to ligand binding to be detected by simple 1D 19F NMR experiments. Interestingly, it was possible to correlate differences in the 19 F chemical shifts changes with the physiological effects of various receptor ligands. In this case, G-protein-activating ligands appear to transmit their effect through one transmembrane helix (helix VI) whereas β-arrestin-biased ligands

predominantly impact the conformation state of helix VII (Figure 1). Further studies used 19F NMR to follow the conversion between active and inactive states in β2AR in response to different ligands binding and allowed the thermodynamics and kinetics associated with these processes to be measured.61 Protein−Membrane Interactions. A number of studies have used 19F NMR to study interactions between peptides or proteins and lipid membranes.2 These interactions are generally difficult to study due to their dynamic and transient nature. However, 19F NMR is uniquely suited to probe such interactions, because changes in the relaxation properties of the nucleus (manifested as line broadening in the spectrum) readily capture dynamic phenomena. Moreover 1D 19F NMR are simple enough that line broadening can be easily quantified, whereas a similar analysis is much harder in a crowded proton spectrum. The actinoporin equinatoxin II (EqTII) is one of a family of sea anemone toxins that disrupts the cell membrane by inducing the formation of pores.29 To elucidate the binding interface of EqTII with lipid membranes, the five Trp residues in the protein, which are thought to interact with membrane lipids, were replaced with 5-fTrp using a Trp auxotrophic strain of E. coli. The structural changes that occurred upon the addition of lipid micelles or bicelles to the protein were followed using 19F NMR. Through these experiments, the interaction and orientation of EqTII with respect to the lipid membrane could be deduced. Our laboratory has used 19F NMR to study the interaction between lipid bilayers and the antimicrobial peptide, MSI-78, C



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Figure 2. Use of 19F NMR to follow the kinetics of protein folding. In this example the slow-folding G121 V mutant of intestinal fatty acid binding protein was labeled with 4-fPhe. (a) The crystal structure (PDB 2IFB) of apo-IFABP with the location of 8 Phe residues shown in blue). (b) The 19F NMR spectrum of fluorinated IFAB (black) and G121 V mutant (red) are overlaid and 19F peak assignments are indicated. (c,d) Stopped-flow (c) and real-time (d) 19F NMR kinetic spectra of 19F labeled G121 V IFBP right after diluting from 6 to 1 M urea at 10 °C. (e) Plot of intensity changes for 4-fPhe-2/17, 4-fPhe-47, and 4-fPhe-62 in short time. (f) Plot of intensity changes of the disappearance of unfolding intermediates (4-fPhe-62i/ 68i), and the appearance of the native folding state for 4-fPhe-62. Images were reproduced and modified with permission from ref 24. Copyright 2007, National Academy of Sciences, U.S.A.

degree of solvent exposure to be assessed. Positions in the hydrophobic core of peptide−membrane complex showed the greatest decrease in mobility and least solvent accessibility, whereas residues that interact with lipid head groups were found to be more mobile and more solvent accessible. Solid-state 19F NMR has been used to study the interaction of antimicrobial peptides with lipid membranes.63,64 Solid-state measurements, on peptides, inserted into mechanically ordered lipid bilayers, allow the orientation of the reporter nucleus relative to the lipid bilayer to be deduced. For example, 4tfmpG was incorporated into PGLa as a probe to provide a rigid connection between the peptide backbone and the 19F reporter.63 It was shown that the peptide adopts three discrete

which exerts its antimicrobial properties by inserting into bacterial membranes to form toroidal pores.40,41,62 A series of MSI-78 peptides that incorporated L-4,4,4-trifluoroethylglycine (tfGly) at different positions in the sequence were synthesized to examine how the structure and dynamics of the peptide change on binding the lipid bilayer. The binding of MSI-78 analogues to lipid bicelles could readily be detected by changes in 19F chemical shift, with large upfield shifts being associated with the most hydrophobic positions in the peptide. Transverse relaxation (R2) measurements of the 19F nucleus, allowed changes in the local mobility of MSI-78 that occur on binding to the lipid bilayer to be measured, whereas solvent isotope effects (H2O/D2O) on the 19F chemical shift allowed the D



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Figure 3. Investigation of oligomeric species during the aggregation of Aβ1−40 using 19F NMR. (a) Time course of Aβ1−40-tfMet35 aggregation followed by 19F NMR. (b) Contour plots showing changes in the 19F spectrum of Aβ1−40-tfMet35 as a function of time (vertical axis). (c) Representative spectrum of Aβ1−40-tfMet35 indicating the major and minor peaks. (d) Scheme describing a possible aggregation pathway for Aβ1−40tfMet35 based on 19F NMR and other experimental data. The peaks corresponding to the various intermediates are marked on the 19F spectrum. The images were reproduced and modified with permission from ref 44. Copyright 2013, American Chemical Society.

resolution of ∼5 s. With this approach, it was possible to identify intermediate states on the pathway to the natively folded protein and obtain rate constants for their formation and disappearance. The first phase, which occurred within a few milliseconds, was the nucleation of structure around a key βturn in the predominantly antiparallel β-sheet protein, which could be detected by chemical shift changes in three Phe residues in the vicinity of the turn. In the second phase, the Nterminal of the protein folded, as revealed by the appearance of native 19F chemical shifts for 4-fPhe-2, 4-fPhe-17, and 4-fPhe 47. The final phase involved the rearrangement of the cterminal region from a non-native collapsed state to the fully native protein, monitored by the 19F chemical shifts of 4-fPhe62, 4-fPhe-68, 4-fPhe-93, and 4-fPhe-128. Interestingly some residues could be observed in multiple conformations during the folding process suggesting fluctuations in structure and/or the presence of a heterogeneous population of intermediates. Protein Aggregation and Misfolding. 19F NMR has increasingly attracted interest as a useful tool to study protein misfolding and aggregation pathways. These phenomena play important roles in human diseases including metabolic diseases such as type II diabetes and neurodegenerative such as Alzheimer’s and Parkinson’s disease.69 Although amyloid plaques typify the pathology of these diseases, the pathways by which the precursor proteins aggregate remain poorly characterized because of the transient and dynamic nature of the intermediate species. Furthermore, there is growing evidence that these intermediates may be more toxic than the

orientations in the lipid bilayer, which interconvert in a temperature-dependent manner, and are also dependent upon the phase (liquid crystalline or gel phase) of the lipid bilayer. Protein Folding. One of the most challenging aspects of protein folding is the detection of transient intermediates in the folding pathway.65 19F NMR of fluorine-labeled proteins has proved a useful tool to study these processes.66 Proteins are typically labeled on aromatic residues, which can readily be accomplished by using a bacterium auxotrophic for the particular amino acid and including fluorinated aromatic amino acid in the grown medium. Such studies, which stretch back over several decades, comprise some of the earliest investigations on fluorinated biomacromolecules. Although 19F NMR does not have either the time resolution or the sensitivity of techniques such as fluorescence or circular dichroism (CD) spectroscopy, it has the advantage of providing simultaneous, position-specific information on protein folding at multiple points in the protein throughout the course of the folding pathway. In this respect it provides an attractive alternative to the technically demanding pulsed H/D exchange experiments that have been used to follow protein folding.67,68 In a recent example, 19F NMR was used to examine intermediates formed during the folding of a slow-folding mutant of rat intestinal fatty acid binding protein (Figure 2).22,24 Substitution of the 8 Phe residues by 4-fPhe (incorporated by using auxotrophic bacteria) provided 8 spectrally distinct probes with which to follow the folding pathway. Using a spectrometer equipped with a stopped-flow mixer it was possible to follow spectral changes with a time E



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broadening of the 19F resonances in the presence of various liposomes and micelles allowed both lipid−protein interactions and protein oligomerization to be assessed. These studies indicate that the fibril-forming path occurs without accumulation of low molecular weight intermediates. The N terminal region of α-synuclein appears to provide the most important membrane-binding interactions leading to an aggregation-prone state. Lastly, a conformational switch involving the central region of the protein results in a transition from the aggregation-prone state to an extensively helical state that binds tightly to micelles. 19 F NMR has also been applied to the study of prion proteins (PrPs). Misfolding of these proteins leads to a range of rare but invariably fatal neurological diseases in humans and animals; for example, Creutzfeldt−Jakob disease and kuru in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep.69,81 In its nonpathological state PrP is associated with neuronal membranes where it monomeric and mainly α-helical. Its misfolded, pathological state (PrPSc) is characterized by a βsheet rich structure that readily aggregates. PrPSc can catalyze the conversion of PrP to PrPSc, a process that forms the basis for the transmission of some of these diseases from one animal to another. A 3-fPhe-labeled variant of PrP has been used to investigate the thermodynamic driving force underlying the interconversion of an early non-native monomeric form of PrP (known as the β-state) to octameric and larger oligomeric forms that are believed to populate the pathway to fibril formation.20 Using 19 F NMR it was possible to quantify the relative populations of monomeric, octameric, and higher oligomers of PrP and determine equilibrium constants for their interconversion. By studying these equilibria as a function of both temperature and pressure, it was possible to determine the enthalpic and entropic contributions to the transitions from monomer to octamer and onward to larger oligomers. Further analysis of the line broadening associated with the NMR peaks allowed estimates of the rates at which the various species interconverted. This study nicely illustrates how detailed thermodynamic information on a complex system can be extracted using 19F NMR as a nonperturbing probe that can report on multiple species simultaneously. In Vivo Studies of Protein Structure. The investigation of protein dynamics and structure in living cells is important for understanding how proteins function under physiological conditions, which can be very different from the conditions employed in vitro studies. H/C/N NMR signals from a protein of interest are hard to observe due to the crowded environment in the cell. Overexpressing the protein of interest and growing the cells on nutrients enriched with NMR active nuclei such as 15 N or 13C can increase the signal; however, this approach does little improve the signal-to-noise ratio because the label is incorporated into other cellular macromolecules. 19F NMR is attractive tool to investigate protein structures and dynamics in cells, because of the high intrinsic sensitivity of 19F nucleus and chemical shift dispersion. Methods employing amber-suppressing tRNAs allow noncanonical amino acids such as tfmPhe and 3-fTyr to be incorporated fairly specifically into proteins that are overexpressed in E. coli. In vivo 19F NMR spectra from globular proteins of up to ∼100 kDa have been recorded,36,37 and analysis of the fluorine chemical shift and line width then allows the structural and dynamic properties of the protein to be evaluated. In contrast, the more commonly used approach of recording 15N−1H

plaques that accumulate as the end products of aggregation, making their characterization all the more important. Our laboratory recently demonstrated the utility of 19F NMR to provide a direct, sensitive and real time measurement of amyloid formation by the highly amyloidogenic peptide, islet amyloid polypeptide (IAPP).70 IAPP is a 37-residue peptide hormone secreted from pancreatic β-cells, along with insulin, that contributes to glycemic control.71 An insoluble fibril form of IAPP is found in the pancreas of up to 90% of patients with type II diabetes that may contribute to the pathology of the disease.72−75 The consumption of monomeric IAPP, that incorporated tfmPhe in place of Phe-23, could be followed directly in real time by 19F NMR. 19F NMR provided convincing evidence that no soluble intermediates accumulate in the aggregation pathway as no new 19F peaks were observed in the spectrum during the time course of aggregation. Furthermore, the rate of monomer consumption, determined by NMR, closely matched the rate of amyloid formation measured by both dye-binding assays and by following changes in secondary structure by CD. Therefore, by combining this information it could be concluded that IAPP fibers form from monomeric IAPP without an appreciable buildup of nonfibrillar intermediates during aggregation, in contrast to most amyloidogenic proteins as described in the following.76 In Alzheimer’s disease, amyloid plaques are largely composed of the Aβ peptide, with plaque formation having been proposed to be the ultimate upstream cause of this disease (amyloid cascade hypothesis).77−79 In contrast to IAPP, numerous oligomeric intermediates of Aβ peptide have been isolated both in vitro and in situ from tissue samples of Alzheimer’s patients. These oligomers may or not be on the pathway to amyloid formation and some oligomeric forms may exhibit significant toxicity of themselves. To study the process of Aβ aggregation using 19F NMR, we synthesized a 19F-labeled Aβ1−40 peptide (Aβ1−40-tfMet35) in which methionine at position 35 was replaced by tfMet to serve as a reporter and the aggregation of the peptide followed over a period of 7 weeks (Figure 3).44 19F NMR detected at least six spectroscopically distinguishable oligomers that form during fiber formation; moreover, the kinetics with each species formed and decayed could be followed. By combining these data with that obtained from electrospray ionization-ion mobility mass spectrometry, CD spectrosocopy, and atomic force microcopy, it was possible to obtain information on size and secondary structure associated with some of these soluble intermediates. The kinetics of Aβ aggregation are very sensitive to temperature, pH, peptide concentration, and buffer composition. Therefore, the ability to follow the formation and decay of multiple species in a single 1D-NMR experiment should provide a valuable tool for understanding these complex aggregation behaviors. α-Synuclein provides another example in which 19F NMR has provided insights into the process of protein misfolding. This 140-residue protein is intrinsically disordered and prone to the formation of amyloid fibrils that are the major components of Lewy bodies associated with Parkinson’s disease.80 The protein is hypothesized to interact with membranes and has been shown to bind to liposomes, which may promote aggregation. Conformational changes associated with membrane binding and fibril formation have been probed by replacing tyrosine residues with 3-fTyr using auxotrophic bacteria31 or tfmPhe using amber-suppressing codons at various positions.34,38 Analysis of the chemical shift changes and lineF



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HSQC experiments (with 15N-enriched proteins) to assess protein folding does not work well in vivo due to excessive signal broadening caused by the viscous nature of the cellular milieu. Because 19F NMR spectra can be acquired in minutes (compared to 1 h or more for 15N−1H HSQC experiments) on proteins at near physiological concentrations, it has the potential to monitor protein folding and/or modifications involved in signal transduction and metabolism in real time experiments.36 In a recent example, 19F NMR was used to investigate whether macromolecular crowding contributes to the stability of proteins folding inside the cell.37 Crowding is predicted to stabilize protein structure because the molecular volume of a folded protein is significantly smaller than that of an unfolded protein and the macromolecular concentration in the cell is extremely high. A marginally stable variant of the immunoglobin G binding domain of Streptococcus protein L was labeled in vivo with 3-fTyr, which replaced three endogenous Tyr residues. Comparison of the 19F NMR spectrum of the fluorinated protein L variant measured in dilute solution in vitro, where it is known to be predominantly unfolded, with that of the protein in cells led to the conclusion that the protein also fails to fold within the cell. The experiments suggested that, surprisingly, stabilizing effects due to macromolecular crowding are insufficient to overcome even relatively small decreases in protein stability. Other nonspecific interactions between cytoplasmic components presumably outweigh crowding effects.

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CONCLUSION Although studies on fluorinated proteins extend over several decades,18,19 advances in NMR spectrometer sensitivity together with the increasing ease and precision with which non-natural amino acids (or fluorinated tags) can be introduced into proteins and peptides has led to a resurgence in the use of 19 F NMR to study a wide variety of biological systems. In particular, a principle advantage of 19F NMR is the exquisite sensitivity of this nucleus to its environment, making it an excellent probe for monitoring subtle conformational changes and transient interactions. As with other nuclei, 19F relaxation measurements, or simple line-width analysis, can provide valuable information on local dynamics of proteins that are increasingly being recognized as essential for biological activity. Lastly, we note that these techniques are relatively simple to implement: many fluorinated amino acids and fluorinated labeling reagents are commercially available and methods for introducing them into proteins and peptides are fairly straightforward. For most studies only simple 1D 19F spectra are needed, and these can easily be recorded on a standard high-field spectrometer equipped with a 1H probe.

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The authors declare no competing financial interest.

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KEYWORDS Amyloid: Insoluble fibers formed by irreversible misfolding and aggregation of proteins and peptides. The fibers characterized by a high β-sheet content and are associated with neuropathies such as Alzheimer’s, Parkinson’s, and Creutzfeldt−Jakob diseases and metabolic diseases such as type II diabetes. Native chemical ligation: A technique for constructing a large polypeptide from two or more unprotected peptides segments. The thiolate of an N-terminal cysteine residue of one peptide attacks a C-terminal thioester of a second peptide. The intermediate thioester bond subsequently rearranges to form the more stable peptide bond resulting the ligation of the two peptides. Nuclear Overhauser Effect (NOE): The transfer of magnetization from one nucleus to another through space. Because the efficiency of transfer is highly distancedependent NOEs can be used to infer the distance between two atoms and hence protein conformations or ligandprotein binding geometries. Relaxation: In the context of NMR this refers to the time taken for the excited nuclear spin to emit a photon and return to the ground state. The rate of relaxation is influenced, among other things, by local motions of the nucleus, making it a useful tool to investigate the mobility of proteins at the level of individual residues.

REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 734 763 6096. E-mail: [email protected]. Present Address §

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States Funding

This work was supported in part by grants from Department of Defense Multidisciplinary University Research Initiative (DoD 59743-CH-MUR) and the Defense Threat Reduction Agency (HDTRA1-11-1-0019) to E.N.G.M. G



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F NMR to Probe Biological Interactions of Proteins and Peptides

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