Site-Specific Tagging of Proteins with Paramagnetic Ions for

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Site-Specific Tagging of Proteins with Paramagnetic Ions for Determination of Protein Structures in Solution and in Cells Xun-Cheng Su* and Jia-Liang Chen

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State Key Laboratory of Elemento-organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China

CONSPECTUS: High-resolution NMR spectroscopy is sensitive to local structural variations and subtle dynamics of biomolecules and is an important technique for studying the structures, dynamics, and interactions of these molecules. Smallmolecule probes, including paramagnetic tags, have been developed for this purpose. Paramagnetic effects manifested in magnetic resonance spectra have long been recognized as valuable tools for chemical analysis of small molecules, and these effects were later applied in the fields of chemical biology and structural biology. However, such applications require the installation of a paramagnetic center in the biomolecules of interest. Paramagnetic metal ions and stable free radicals are the most widely used paramagnetic probes for biological magnetic resonance spectroscopy, and therefore mild, high-yielding approaches for chemically attaching paramagnetic tags to biomolecules are in high demand. In this Account, we begin by discussing paramagnetic species, especially transition metal ions and lanthanide ions, that are suitable for NMR and EPR studies, particularly for in-cell applications. Thereafter, we describe approaches for site-specific tagging of proteins with paramagnetic ions and discuss considerations involved in designing high-quality paramagnetic tags, including the strength of the binding between the metal-chelating moiety and the paramagnetic ion, the chemical stability, and the flexibility of the tether between the paramagnetic tag and the target protein. The flexibility of a tag correlates strongly with the averaging of paramagnetic effects observed in NMR spectra, and we describe methods for increasing tag rigidity and applications of such tags in biological systems. We also describe specific applications of established site-specific tagging approaches and newly developed paramagnetic tags for the elucidation of protein structures and dynamics at atomic resolution both in solution and in cells. First, we describe the determination of the 3D structure of a short-lived, low-abundance enzyme intermediate complex in real time by using pseudocontact shifts as structural restraints. Second, we demonstrate the utility of stable paramagnetic tags for determining 3D structures of proteins in live cells, and pseudocontact shifts are shown to be valuable structural restraints for in-cell protein analysis. Third, we show that a NMR optimized paramagnetic tag allows one to determine distance restraints on proteins by double electron−electron resonance (DEER) measurements with high spatial resolution both in vitro and in cells. Finally, we summarize recent advances in site-specific tagging of proteins to achieve atomic-resolution information about structural changes of proteins, and the advantages and challenges of magnetic resonance spectroscopy in biological systems.

1. INTRODUCTION Proteins play central roles in living systems, and elucidation of their structures, dynamics, and interactions is crucial for understanding their function. Recent progress in this area has resulted from advances in biophysical methods in the fields of cell biology, structural biology, and chemical biology. Many biophysical methods rely on modification of proteins with a functional group, such as a fluorophore, a mass marker, or a magnetic group, which provides a handle for analysis by a spectrometric or imaging technique. For example, site-specific © XXXX American Chemical Society

labeling of proteins with a paramagnetic tag and analysis by NMR can provide a trove of structural information.1 Observation of paramagnetic effects manifested in NMR spectra is an effective way to elucidate the structures, dynamics, and interactions of biomolecules,1−4 and paramagnetic tags play central roles in generating reliable structural and dynamic restraints for biomolecules. Many proteins do not have a Received: March 10, 2019

A

DOI: 10.1021/acs.accounts.9b00132 Acc. Chem. Res. XXXX, XXX, XXX−XXX

Article

Accounts of Chemical Research

Scheme 1. (A) Schematic Illustration of Site-Specific Labeling of a Protein with a Paramagnetic Tag (red sphere) by Means of a Reaction between a Reactive Thiol Group on the Protein and the Tag (blue) and (B) Reaction Strategies as Described in Part A for Labeling of Proteins with Paramagnetic Tags Comprising a Paramagnetic Metal and a Chelating Moiety

have been extensively reviewed.1−4 Both PCSs and contact shifts are caused by electron−nuclear dipolar interactions. Contact shifts occur only for nuclear spins that are within five covalent bond lengths (∼5 Å) of the paramagnetic center. PCSs provide long-range structural restraints of proteins and can be determined readily and accurately by comparison of the chemical shifts in the NMR spectra of paramagnetic and diamagnetic species. Metal ions with unpaired electrons are potential candidates for NMR and EPR applications in biological systems. Cr3+, Mn2+, Cu2+, and Gd3+ are paramagnetic but show little or no magnetic anisotropy, and they are used for PRE measurements. Because of its PRE effects, Ni2+ has been used to assess the solvent-exposed surfaces of proteins by means of NMR,10 but the paramagnetism of Ni2+ depends on its coordination complex. Small PCSs have been observed when some proteins are titrated with Ni2+,11 but a strongly chelating ligand produces a diamagnetic Ni2+ complex.12 High-spin Fe2+/Fe3+ complexes are paramagnetic and are well-characterized in heme-binding proteins, but low-spin Fe2+ is diamagnetic. Some

paramagnetic center and methods for site-specifically labeling proteins with a paramagnetic motif are generally required for the use of paramagnetic NMR and also for electron paramagnetic resonance (EPR) spectroscopy.5,6 In this Account, we outline recent progress in the development of methods for chemically tagging proteins for NMR and EPR studies, and we describe some applications of the tagged proteins in solution and in cells.

2. PARAMAGNETIC TAGGING OF PROTEINS 2.1. Lanthanide Ions for in Solution and in Cell Biological Magnetic Resonance

Dipolar interactions of unpaired electrons and nuclear spins markedly affect the relaxation of nuclear spins as well as the chemical shifts that are measurable by NMR.1−4 The paramagnetic effects caused by dipolar interactions between unpaired electrons and nuclear spins in NMR spectra generally fall into five categories: paramagnetic relaxation enhancement (PRE), pseudocontact shifts (PCSs), contact shifts, residual dipolar couplings (RDCs), and cross-correlation relaxation (CCR).7−9 These paramagnetic effects in structural biology B


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Figure 1. Structures of chelating agents that can be attached to proteins via disulfide bond formation.

high-spin Co2+ complexes have magnetic anisotropy similar to that of Yb3+ and are suitable for protein analysis by NMR.12−15 Recent progress on double electron−electron resonance (DEER) by using nitroxide radicals, trityl radicals, Mn(II), and Gd(III) demonstrate great potential in determining distance restraints between paramagnetic centers that were attached to biomolecules.5,6,16−21 For in-cell applications of NMR and EPR in particular, only paramagnetic species that meet stringent requirements with regard to their thermostability, kinetic properties, and redox stability are suitable. The advantages of using paramagnetic lanthanide ions in biological systems arise from the diversity of their magnetic anisotropies for measurement by NMR spectroscopy.1−3,7,22 The Gd3+ complex is magnetic and isotropic and shares long electron relaxation time and therefore can be applied as a PRE probe for biomolecular NMR study. The Gd3+ spin label attests to the high quality of Gd3+ as a tag for DEER measurements.16 It is noted that lanthanides are not physiologically relevant elements in most living systems, so there are no background signals from the cytoplasm, and most trivalent lanthanide ions are redox stable. The redox stability of paramagnetic lanthanide ion complexes in cells is beneficial for NMR and EPR spectroscopy. Lanthanide ions are chemically similar to one another, but these ions show diverse magnetic properties depending on the number of 4f electrons (4f0−4f14). Lanthanide ions are usually regarded as hard acids (according to Pearson’s scheme) and have high coordination numbers, 8 or 9. Y3+ has chemical properties similar to those of lanthanide ions, and its ion radius (1.04 Å) sits in the middle of the range between that of La3+ (1.10 Å) and that of Lu3+ (1.00 Å).23 Therefore, Y3+, La3+, and Lu3+ serve as excellent diamagnetic references for the determination of PCSs and PREs generated by paramagnetic lanthanide ions. In contrast to the case for small molecules, in biological systems, contact shifts similar to the PCSs produced by paramagnetic ions are negligible because the nuclear spins of biomolecules are far from the paramagnetic center, and therefore reliable and accurate PCSs can be determined. Lanthanide ions have high coordination numbers, and therefore a chelating moiety containing multiple carboxylate groups or a DOTA-like compound (DOTA = 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid, introduced by

the Ubbink group and the Otting group into proteins) is needed for the formation of stable metal complexes.1,3 Because NMR spectroscopy is sensitive to magnetic anisotropy, it is preferable to avoid forming new chiral centers upon coordination of a paramagnetic lanthanide ion when designing paramagnetic tags. 2.2. Site-Specific Labeling of Proteins with Paramagnetic Tags

Strategies for site-specific labeling of proteins with paramagnetic tags for biomolecular NMR spectroscopy have been discussed in several reviews.1,3,24,25 Paramagnetic tags generally contain two main functional moieties, a reactive group for reaction to a specific site in a protein and a metal chelator to immobilize the paramagnetic center (Scheme 1A). The protein tagging reaction has to be mild, site-specific, and efficient so that the three-dimensional structure and the activity of the target are maintained. The tagging yield has to be high, especially for isotope-labeled samples used for NMR. Cysteine is one of the least abundant amino acids in proteins; we therefore focus on the use of thiol-selective 4-phenylsulfone pyridine as a versatile building block for anchoring rigid and stable paramagnetic tags site-specifically in proteins. 2.3. Site-Specific Labeling of Proteins via Disulfide Bond Formation

Disulfide bond formation is the most widely used strategy for labeling proteins with paramagnetic tags,1 and this ligation method has several advantages. For one thing, the tagging reaction is efficient and high-yielding (Scheme 1B, routes 1− 3). As shown in Figure 1, a number of tags containing a metalchelating moiety and an active thiol group for attachment to proteins have been synthesized and subsequently evaluated by NMR spectroscopy. For example, L-Cys−DTPA has been conjugated with 15N-ubiquitin, and when the conjugate is complexed with a paramagnetic lanthanide ion, the 15N heteronuclear single-quantum correlation (HSQC) NMR spectrum shows one paramagnetic species, and large PCSs are observed.26 In contrast, over two paramagnetic species are observed for the same lanthanide complex in a ubiquitin−LCys−EDTA conjugate.26 4MMPyMTA and 4MPyMTA have been tested with several proteins, and both tags produce large PCSs.27,28 The fluorescent paramagnetic tags 4MTDA and C


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Figure 2. Structures of chelating agents containing vinylpyridine or phenylsulfone pyridine moieties.

two reagents, strategies involving the formation of thioether bonds between tags and the thiol groups of proteins via the reactions of vinylpyridines (Scheme 1B, route 6) and 4phenylsulfonated pyridines (route 7) have been proposed. Both strategies rely on the electron-withdrawing effects of the pyridine moiety on a thiol−ene addition reaction in the first case and on a nucleophilic substitution reaction in the second case. In addition, coordination of pyridine nitrogen with a paramagnetic metal ion restricts movement of the paramagnetic center. 2.4.1. Radical-Free Thiol−Ene Reactions for SiteSpecific Tagging of Proteins. 4-Vinylpyridine was reported to quantify thiol abundance in a reduced, denatured protein.33 In addition, we showed that reaction of vinyl-substituted DPA (Figure 2) with solvent-exposed cysteine under mild conditions can be used for site-specific tagging of proteins.32 The electron-withdrawing nature of the pyridine ring permits the thiol−ene reactions between the vinyl group and solventexposed cysteines to proceed smoothly in aqueous solution without the need for radical initiation. In addition, no side reactions between the vinylpyridines and protein amino

4MMTDA form stable lanthanide complexes in aqueous solution.29 Formation of disulfide bond tethers between proteins and paramagnetic tags is efficient in paramagnetic labeling of proteins, but the disulfide bond is unstable in reducing environments and susceptible to thiol−disulfide exchange reactions at high pH. A more stable linker between proteins and paramagnetic tags is necessary for applications, especially in cells. 2.4. Site-Specific Labeling of Proteins via C−S Bond Formation

Maleimide and acetylhalo derivatives (Scheme 1B, routes 4 and 5, respectively) are the most often used reagents for generating stable C−S bonds between proteins and tags.30,31 However, the reaction between a maleimide and a cysteine produces a new chiral center, and as a result, two paramagnetic species are observed in the NMR spectra of the resulting protein conjugate.32 The solution stability and selectivity of acetylhalo derivatives are not completely satisfactory. Therefore, with the goal of circumventing the problems with these D


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suitable for NMR analysis under crowded conditions, owing to the high stability of their complexes with lanthanide ions and the rigidity of the short thioether tether.41 The protein thiol reactivity of 4PS-PyMTA is greater than that of 4V-PyMTA. Notably, when complexed with a paramagnetic lanthanide ion, a 4PS-PyMTA−ubiquitin G47C conjugate generates substantially larger PCSs than the corresponding 4V-PyMTA conjugate (Figure 3). The

groups, either at the N-terminus or on lysine side chains, are observed. The resulting 4V-DPA−protein conjugates are stable in the presence of reducing agents such as tris(2-carboxyethyl)phosphine or dithiothreitol. To increase the number of coordinating sites and thus the binding affinity for lanthanide ions, we designed and synthesized 4V-PyMTA (Figure 2), which reacts with solvent-exposed cysteines at pH 8 to yield protein conjugates that can be used to monitor the in situ structural variations of proteins, and thus the cellular environment, by NMR.34 It was shown that a 4V-PyMTAlabeled peptide was suitable for DEER measurements in cells.18 These derivatives show diverse reactivity toward cysteine thiol groups in aqueous solution. In the thiol−ene reaction at pH 7.5, the reactivity of the tested compounds decreases in the order 4V-DPA > 4V-6M-PyEDTA > 4V-PyEDTA > 4V-TDA > 4V-PyMTA, and the reactivity of 4V-DPA is 200 times that of 4V-PyMTA.35 Introducing a bromine atom at the 3-position of the pyridine greatly enhances reactivity toward protein thiols, as demonstrated by 4V-BrPyMTA. In another study, we demonstrated that 2V-8HQ shows high performance in ubiquitin conjugates. Coordination of a His side chain to Co2+, Ni2+, or Zn2+ is preferable to coordination of Glu or Gln side chain, as indicated by the fact that the association constants of ubiquitin−8HQ with these metal ions are approximately an order of magnitude greater in a ubiquitin E24H/A28C−8HQ conjugate than in an A28C−8HQ conjugate.12 2.4.2. 4-Phenylsulfonated Pyridine Derivatives for Site-Specific Modification of Protein Thiol Groups. Tagging reactions between vinylpyridine derivatives and a cysteine residue generate a conjugate with a flexible linker (comprising two methylene groups) that averages paramagnetic effects, and the experimentally determined magnetic anisotropy tensors are small.34 One effective way to increase the rigidity of a paramagnetic tag is to shorten the tether between the tag and the target protein (Scheme 1B, route 7). This approach originated from an unexpected reaction between 4Br-PyMTA and sodium benzenesulfonothioate in acetonitrile, which produces phenylsulfonated PyMTA, along with a yellow sulfur precipitate, instead of S-(PyMTA)benzenesulfonothioate. When we tested reactions of phenylsulfonated pyridine derivatives with L-cysteine, we were surprised to find that the phenylsulfone group at the 4position of the pyridine is a thiol-specific leaving group.36 That is, 4PS-PyMTA reacts with cysteine quantitatively at pH 6−8, resulting in a thioether bond connecting PyMTA with cysteine. During the reaction, the sulfinate and thioether bridged products were characterized by NMR.36 No reactions of the phenylsulfonated pyridines with the amino groups of free amino acids, with the N-termini of proteins, or with lysine side chains were observed. The overall mechanism of the reactions of phenylsulfonated pyridines with protein thiols is found to be similar to that of the reactions of methylsulfonyl benzothiazole with free thiol groups.37,38 We therefore designed a number of pyridine tags containing a phenylsulfone group (Figure 2), and their performance as site-specific protein tags was evaluated; subsequent follow-up studies were carried out by Müntener et al.39 We synthesized 4PS-PyEDTA and 4PS-6M-PyEDTA (Figure 2) and used these tags to generate Mn(II) complexes of ubiquitin T22C/G47C conjugates for EPR measurements.40 In addition, diethylene triamine pentaacetic acid derivatives 4PS-PyDTTA and 4PS-6M-PyDTTA were found to be

Figure 3. (A) Superimposition of 15N-HSQC spectra recorded for a 0.1 mM ubiquitin G47C labeled with 4V-PyMTA in complex with 0.1 mM Y3+ (red) and 0.05 mM Tb3+ (black). (B) Superimposition of 15 N-HSQC spectra recorded for a 0.1 mM ubiquitin G47C labeled with 4PS-PyMTA in complex with 0.1 mM Y3+ (red) and 0.1 mM Tb3+ (black). The NMR spectra were recorded with a proton frequency of 600 MHz.

determined Δχax component of the magnetic anisotropy tensor for the 4PS-PyMTA−ubiquitin conjugate is 6 times the tensors for 4V-PyMTA,36 implying that removing the two methylene groups in the linker between the protein and tag greatly increases the rigidity of the protein−tag conjugates. Similarly, phenylsulfonated pyridine moieties were attached to a 1,4,7,10-tetraazacyclodecane (cyclen) ring to afford PSPyDO3MA-Ln and PSPy-6M-DO3MA-Ln (Figure 2). The concept behind these complexes was based on the crystal structures of DO3MA−Gd42 and DO3A-Py−Gd43 complexes: coordination of pyridine with a lanthanide restricts the flexibility of the pyridine linker, and the presence of the methyl group in the acetate arm inhibits rotation of the arm in the lanthanide complex. The reaction of pyridyl nitro substituents containing the DOTA-Ln moiety with solvent exposed cysteines, in generation of stable thioether linker was proposed by the Parker group.21,44 We found that the pyridyl nitro group is sensitive to reducing reagents like TCEP in the protein labeling reactions (data not shown). Both PSPy-DO3MA-Ln and PSPy-6M-DO3MA-Ln produce large PCSs in protein conjugates,45 and only one major paramagnetic species is observed. However, the protein ligation reactions of PSPy-DO3MA-Ln are slow, and high pH (∼9) is required for the tagging reactions. E


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Accounts of Chemical Research The additional methyl group in 4V-6M-PyEDTA, 4PS-6MPyEDTA, 4PS-6M-PyDTTA, and PSPy-6M-DO3MA-Ln (Figure 2) increases the reactivity of the tag with protein thiols. In addition, the presence of the 6-methyl group on the pyridine ring restricts the flexibility of the protein−tag−Ln complex, resulting in larger Δχ tensors determined by NMR spectroscopy41,45 and narrower distance distributions determined by EPR spectroscopy.40 One disadvantage is that introduction of a methyl group decreases the overall metal binding affinity.40,41 To increase the reactivity of the DOTA-like tags containing a thiol-specific phenylsulfone pyridine moiety, we designed tags with a 3-Br or 5-Br substituent on the pyridine ring: BrPSPy-DO3MA-Ln and BrPSPy-DO3A-Ln. As expected, both of these tags show greater reactivity than PSPyDO3MA-Ln and PSPy-6M-DO3MA-Ln: their ligation reactions with protein thiols are generally complete within 8 h at room temperature in aqueous solution at pH 8. In addition, the protein−BrPy-DO3MA-Ln complexes with Tm3+ and Yb3+ exist as one predominant paramagnetic species in solution by NMR.46 The protein−BrPy-DO3A-Ln (Dy3+ or Tm3+) produces two major paramagnetic species in NMR spectra (data not shown), but protein−BrPy-DO3A-Gd demonstrates higher performance in DEER measurement in increased sensitivity and longer phase memory time with similar distance resolution.47

Figure 4. (A) Schematic illustration of the factors that determine the flexibility of protein−tag conjugates: the stability and flexibility of the linker (1), the thermal and kinetic stability of the paramagnetic tag (2), and ligation site in a protein in the designing of paramagnetic tags. (B) Comparison of the sensitivity of RDCs (using the backbone N−H bond) and PCSs (drawn with PCS isosurface) with respect to the movement of the paramagnetic center as installed in Scheme 1A.

2.5. Flexibility of Paramagnetic Tags in Protein Conjugates

Increasing attention is being paid to the flexibility of paramagnetic tags used for the characterization of dynamic biomolecular systems, because rigidity is a critical factor in determining the quality of paramagnetic effects. PCSs and RDCs, produced by magnetic anisotropy, are sensitive to the flexibility of a paramagnetic center, and fast exchange averaging will decrease the magnitude of PCSs and RDCs.1−3,48,49 Key determinants of the flexibility of a protein−tag conjugate include the length and rigidity of the linker between the protein and the tag, the charge and size of the tag, and lability of metal−tag complex, bond rotation in the chelating moiety, and also the ligation site in a protein (Figure 4A). The determined PCS (in ppm) in a protein−tag complex can be described as in eq 1:1,2,8 PCS =

A ax,rh =

(2)

where Aax,rh are the axial and rhombic components of the alignment tensor determined from the RDC values that can be determined by fitting the RDC values to the structure of protein and Δχax,rh are the axial and rhombic components determined from the PCSs as shown in eq 1. The difference in sensitivity between the PCSs and RDCs to protein mobility was first recognized in a cytochrome C K77A mutant at high pH.49 The sensitivity of PCSs to the movement of the paramagnetic center decreases as the distance of the nuclear spin from the paramagnetic center increases. In contrast, RDCs do not depend on distance from the paramagnetic center but are instead sensitive to the reorientation of the N−H vector with respect to the principle axis of magnetic anisotropy (Figure 4B). The comparison between PCSs and RDCs determined in a protein−tag complex is shown in Table 1. In general, the experimentally determined Aax values from RDC data are generally smaller than the back-calculated data using eq 2, which Δχax are determined from PCS data. It is noted that in the case of extensive mobility, an averaged tensor might not exist to fit the data.2

1 [Δχax (3 cos 2 θ − 1) + 1.5Δχrh sin 2 θ 12πr 3 cos 2ϕ]

B0 2 Δχ 15kTμ0 ax,rh

(1)

where r, θ, and ϕ are the polar coordinates of the nuclear spin with respect to the principal axes of the magnetic susceptibility anisotropy tensor and Δχax and Δχrh are the axial and rhombic components of the magnetic anisotropy tensor. The detailed definition of magnetic susceptibility anisotropy tensor can be found in excellent reviews.1,2,8 The tensor parameters can be determined by fitting the PCS values to the structure of the protein, which was site-specifically labeled with a paramagnetic metal ion (as shown in eq 2). The position of the metal, sitespecifically attached to a protein via covalent modifications (Scheme 1A), must be determined accurately, but this is difficult to achieve. In addition, the rigidity of the paramagnetic tag cannot be simply evaluated from the determined Δχax and Δχrh values. One way to evaluate the flexibility of a paramagnetic tag is to compare the correlations between tensor components determined from PCSs and RDCs,1,3,8 both produced by the same paramagnetic ion:

2.6. Comparison of Magnetic Anisotropies of Lanthanide Ions Complexed with Linear and Cyclic Chelators

We compare the magnetic anisotropy determined for lanthanide ions complexed with linear and cyclic chelating ligands in the protein conjugates. Linear tags generally have an open backbone chain moiety including a polycarboxylate ligand, a lanthanide-binding peptide, or a metal-binding motif in a protein that forms stable complexes with lanthanide ions as an open linear tag. Cyclic ligands, such as the DOTA-like F


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formed readily by means of stepwise titration. In contrast, complexes between protein−DOTA-like conjugates and lanthanides have to be prepared by ligation of the protein to the DOTA-like−lanthanide complex, and a variety of tagging reactions with different DOTA-like lanthanide ion complexes are invariably required. The magnitudes of the magnetic anisotropies of linear tag complexes were generally in agreement with Bleaney’s theory.50,51 However, discrepancies between the determined magnetic anisotropy tensors were observed when a given lanthanide was in complex with a linear or cyclic tag attached to a protein.45,52 Specifically, when a ubiquitin G47C mutant was ligated to 4PS-PyMTA or PSPyDO3MA-Ln, large variations were observed in the determined paramagnetic Δχ values and alignment tensor values (determined from the PCSs and RDCs, respectively) (Table 1). Similar variation was observed for a GB1 T11C mutant labeled with PSPy-6M-DO3MA-Tm and 4PS-PyMTA.36,45,52 In general, DOTA-like lanthanide complexes produce greater magnetic anisotropy. The mechanism for these differences deserves further consideration. Mironov et al. proposed that the coordination geometry of DOTA-like lanthanide complexes also influences the paramagnetic anisotropy,53 and Funk et al. have observed cases that disobey Bleaney’s prediction.54 The local environment at the tagging site also influences the determined paramagnetic anisotropy, and it is advisible to take into account the local structural variations in evaluation of paramagnetic effects.

Table 1. Comparison of Magnetic Anisotropies Generated by Paramagnetic Lanthanide Ions Complexed with Ubiquitin G47C and GB1 T11C Conjugated with Various Tagsa Tb3+

Dy3+

Tm3+

Yb3+

34

Δχax Δχrh Δχax Δχrh Aax Arh Δχax Δχrh Aax Arh Δχax Δχrh Δχax Δχrh

Ubi G47C−4V-PyMTA 4.3 1.7 2.3 1.1 Ubi G47C−4PS-PyMTA36 −21.5 −24.5 16.6 −5.1 −7.1 3.9 1.4 (−5.5) 1.6 (−6.3) −1.2 (4.2) 0.2 (−1.3) 0.2 (−1.8) −0.3 (1.0) Ubi G47C−PSPy-6M-DO3MA-Ln45 −84.3 65.2 −17.2 31.8 −6.2 (−21.6) 4.2 (16.7) −4.2 (−4.4) 1.8 (8.1) GB1 T11C−4PS-PyMTA52 6.1 3.4 −2.9 3.6 1.9 −0.8 GB1 T11C−PSPy-6M-DO3MA-Ln45 51.2 22.1 20.3 12.1 3.4 0.9

0.9 0.4 6.7 1.4 −0.4 (1.7) −0.1 (0.4)

a Δχax (10−32 m3) and Δχrh (10−32 m3) are the axial and rhombic components of the magnetic susceptibility anisotropy tensor determined from PCSs of the backbone amide protons. Aax (10−4) and Arh (10−4) are the axial and rhombic components of alignment tensors determined from the backbone amide RDCs, and the backcalculated Aax (10−4) and Arh (10−4) from eq 2 are shown in parentheses. All NMR experiments were performed with a proton frequency of 600 MHz at 298 K.

3. DETERMINATION OF PROTEIN STRUCTURES IN SOLUTION AND IN CELLS Having described various protein ligation strategies and the development of functional tags, we next discuss several applications in biological systems including determination of the 3D structure of a short-lived, low-abundance protein complex in real time, determination of protein structure in live

ligands, form kinetically inert and highly stable complexes with lanthanide ions. Lanthanide complexes with linear ligands form rapidly, and lanthanide complexes with protein−tag conjugates can be

Figure 5. (A) Representative NMR spectra of a short-lived, low abundance protein complex (shown in blue) obtained in real time, showing that the small NMR cross-peaks decay gradually with reaction time. (B) Strategy for determining the 3D structure of a short-lived, low-abundance protein complex using PCSs. Panel B was reproduced with permission from ref 28. Copyright 2016 John Wiley and Sons. G


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Scheme 2. Strategy for Determining the 3D Structure of Proteins in Live Cells by Means of Paramagnetic NMR with PCSs as Structural Restraints

and interactions in cellular environments at atomic resolution is highly desirable for understanding processes in living cells. Although NMR spectroscopy has been shown to be a powerful technique for this purpose,59−62 the crowded conditions inside cells generally produce nonspecific associations between proteins and between proteins and other cellular components, and these associations broaden NMR signals. One challenge in recording nuclear Overhauser effect structural restraints in live cells is the need to suppress the strong background signals of cellular components. Development of fast, efficient methods to obtain structural or dynamic restraints for proteins in cells is highly necessary. It can be expected that measurement of in-cell PCSs would be suitable structural restraints for characterizing proteins inside cells (Scheme 2). The strategy includes the following steps: site-specifically labeling a protein with a paramagnetic tag, preparation of a number of paramagnetic protein−tag complexes in vitro, and delivery of the protein−tag samples into live cells followed by collection of PCSs. We sitespecifically attached 4PS-PyMTA (Figure 2) to GB1 T11C mutant and to a GB1 V21C mutant, and 15N HSQC spectra of the GB1−PyMTA complexed with paramagnetic lanthanide ions (Tm3+ and Tb3+) were measured in Xenopus laevis oocytes (Figure 6A). In this way, 15N HSQC spectra with decent signal-to-noise ratio could be recorded within a couple of hours at a protein concentration of ∼30 μM in live cells. Therefore, PCSs of backbone amide protons were collected for the complex of Tm3+ and Tb3+, respectively. Then, using the GPSRosetta program,63,64 we determined the solution structure of GB1 in cells with PCSs as the only structural restraints (Figure 6B).52 This result suggests that PCSs can be used as efficient structural restraints for in cell NMR analysis. Similarly, Müntener et al. reported the preparation of GB1 modified with a DOTA-like tag and determined the in cell structure of the protein by means of GPS-Rosetta program.39

cells by NMR spectroscopy, and the use of rigid paramagnetic tags for high-resolution DEER spectroscopy. 3.1. Determination of the 3D Structure of a Short-Lived, Low-Abundance Protein Complex in Real Time

Characterization of the structure of transient protein complexes by means of current biophysical methods remains a challenge, especially for low-abundance, unstable intermediate states in solution.4 For example, Staphylococcus aureus transpeptidase SrtA forms an unstable intermediate complex with its substrate in solution, as indicated by NMR spectroscopy.28 The unstable thioester complex, formed by reaction of the C184 residue with the LPXTG substrate, disappears gradually from solution within a couple of hours. The maximum concentration of the intermediate complex is only ∼30 μM, as indicated by an 15N HSQC experiment (Figure 5A). For determination of the 3D structure of this complex, PCSs, readily measured in short time, are the only possible structural restraints, because other routinely used restraints (e.g., nuclear Overhauser effects, PREs, and RDCs) are difficult to obtain at concentrations of

Site-Specific Tagging of Proteins with Paramagnetic Ions for

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