CHAPTER SIXTEEN
Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins: A Guide to Spin Label Paramagnetic Relaxation Enhancement Restraints Linda Columbus*,1, Brett Kroncke† *Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA † Department of Biochemistry and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Site-Directed Spin Labeling 1.1 Nitroxide dynamics 1.2 Choice of spin label 1.3 Protein topology and PRE restraint coverage 1.4 Structure and function perturbation 1.5 Labeling efficiency 1.6 Spin label contaminant 2. PRE Measurements 2.1 The reference spectrum 2.2 Calculation of distances from intensity ratios 2.3 Distance classifications 3. Structure Calculation with PRE Restraints 3.1 The nitroxide spin label 3.2 Error bounds for PRE distances 3.3 Planar bilayer restraints 4. Assessment of Structure Quality 5. Future Developments 5.1 Molecular dynamics, explicit solvent, and the native bilayer 5.2 Carbon PREs 6. Summary References
Methods in Enzymology, Volume 557 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.12.005
#
2015 Elsevier Inc. All rights reserved.
332 333 333 334 335 335 336 336 336 337 338 338 338 341 341 341 342 342 343 343 343
329
330
Linda Columbus and Brett Kroncke
Abstract Solution nuclear magnetic resonance structures of polytopic α-helical membrane proteins require additional restraints beyond the traditional Nuclear Overhauser Effect (NOE) restraints. Several methods have been developed and this review focuses on paramagnetic relaxation enhancement (PRE). Important aspects of spin labeling, PRE measurements, structure calculations, and structural quality are discussed.
Polytopic α-helical membrane proteins are challenging systems for solution nuclear magnetic resonance (NMR) approaches. Four major bottlenecks are (i) membrane protein expression in sufficient yields required for in vitro investigations; (ii) extraction, solubilization, and stabilization of folded and functional membrane proteins in membrane mimics such as detergents; (iii) structural heterogeneity and dynamics that are integral to membrane protein function, but interfere with in vitro structural investigations; and (iv) Nuclear Overhauser Effect restraints (NOEs)—the workhorse of NMR structure calculations—do not provide tertiary structural information for deuterated α-helical membrane proteins. For the most recent reviews of challenges i–iii, readers should seek these references for expression (Guerfal et al., 2013; Gupta, Kueppers, & Schmitt, 2014; Kimura-Soyema, Shirouzu, & Yokoyama, 2014; Parker & Newstead, 2014; Su, Si, Baker, & Berger, 2013), solubilization (Columbus et al., 2009; Duquesne & Sturgis, 2010; Schimerlik, 2001), and structural heterogeneity (Cafiso, 2014; Pan, Piyadasa, O’Neil, & Konermann, 2012). There are several methods (e.g., residual dipolar couplings (RDCs) and methyl NOEs) developed to tackle the lack of NOE-based structural restraints and this review will focus on nitroxide spin label paramagnetic relaxation enhancement (PRE)-derived restraints as applied to polytopic α-helical membrane proteins. PRE measurements rely on the incorporation of a paramagnetic species site specifically (e.g., a nitroxide spin label), which enhances the relaxation of neighboring NMR active nuclei in a distance-dependent manner. PRE-proton-derived ˚ ) compared to the standard 1H–1H NOE distances are long range (15–25 A (5–6 A˚) and were used in the solution NMR structure determination of eight (Berardi, Shih, Harrison, & Chou, 2011; Eichmann et al., 2014; Maslennikov et al., 2010; Reckel et al., 2011; Van Horn et al., 2009; Zhou et al., 2008) of the nine polytopic α-helical membrane proteins determined to date (Table 1; with sensory rhodopsin the exception (Gautier & Nietlispach, 2012)). The general workflow for utilizing PRE restraints for the NMR structure determination of α-helical membrane proteins is shown in Fig. 1 and is divided into
Table 1 Solution NMR structures of polytopic integral membrane proteins Restraints in addition to backbone NOEsa PDB IDs
SL-PRE
RDCs
Inter-NOEsb
39
DsbB
2k73, 2k74 1144
337
DAGK
2kdc
67
208
Sensory rhodopsin 2ksy
1536
# of TMa Oligomeric state Year
X-ray structure rmsd (Å)
4
Monomer 2008 2zuq
2.4
3
Trimer
d
7
Monomer 2010 1h68
2009 3ze3
KdpD
2ksf
845
4
Monomer 2010 None
ArcB
2ksd
291
2
Monomer 2010 None
QseC
2kse
295
2
Monomer 2010 None
Proteorhodopsin
2l6x
1006
81
7
Monomer 2011 4jq6c
UCP2
2lck
452
470
6
Monomer 2011 None
YgaP
2mpn
8
2
Dimer
a
87
216
Per monomer. Nonsequential and long range (j1 jj 5). c Protein with 57% identity. d The NMR structure has domain swapping and the crystal structure does not. b
2014 None
1.06
3.71
332
Linda Columbus and Brett Kroncke
■
Introduce unique individual cysteine residues on each TM segment
Choose sites that are in ordered regions of the protein
■
SDSL
Assess the dynamics of the label using CW EPR lineshape analysis
Spin label each site with MTSSL and the diamagnetic equivalent
■
Measure labeling efficiency
■
Determine labels do not influence the global fold
Record the HSQC of each MTSSL-labeled site
PRE measurement
Record the HSQC of each diamagnetically labeled site
■
Normalize peak intensities to account for differences in protein concentration
Measure intensity ratios for each assigned resonance Bin the intensity ratios into three classes
Structure calculation
Combine PRE, RDC, methyl NOE, sequential NOE, hydrogen bonding restraints with chemical shift-derived dihedral angles
■
Model the nitroxide spin label at each position labeled and use the c1 – c3 dihedral restraints for the nitroxide side chain
Calculate ~300 initial structures
■
Use a ±4 Å error bounds on the PRE distance
Select the 20 lowest energy structures
Structure analysis
Comparison of calculated chemical shift from ensemble to observed chemical shifts
rmsd of ensemble
Analysis of helix packing: interhelical crossing angles, interhelical distances, and helical kinks
Figure 1 Workflow diagram for utilizing PRE restraints for the NMR structure determination of α-helical membrane proteins. There are four steps: (i) site-directed spin labeling (SDSL), (ii) the PRE measurement, (iii) the structure calculation, and (iv) the evaluation of the accuracy and precision of the structure. Tips are also provided in the text on the right.
four stages: (i) site-directed spin labeling (SDSL), (ii) PRE measurement, (iii) structure calculation, and (iv) accuracy and precision evaluation of the structure.
1. SITE-DIRECTED SPIN LABELING Typically, SDSL is used to introduce a nitroxide spin label at a unique cysteine residue. Mutagenesis introduces alanine or serine in place of native cysteine residues such that a single cysteine can be introduced throughout
Solution NMR Structure Determination
333
the α-helical membrane protein. Selection of where to place the cysteine residue, and subsequently the spin label, requires a few important considerations.
1.1 Nitroxide dynamics The dynamics of the spin label, which is determined by internal side-chain motions, backbone fluctuations, and rigid body motions, can contribute to large ranges of distances between the label and the relaxed nuclei. However, the PRE measurement is most influenced by the closer distances; the distance range interpreted may only capture a subpopulation of states sampled. Therefore, placing the spin label in dynamic regions may complicate the interpretation of the structure calculated and is best avoided by placing the spin label in structured regions. The dynamics of the spin label, which reflect both the internal flexibility of the spin label and the flexibility of the protein backbone, should always be evaluated using CW EPR (Columbus, Kalai, Jeko, Hideg, & Hubbell, 2001; Hubbell, McHaourab, Altenbach, & Lietzow, 1996; Kroncke, Horanyi, & Columbus, 2010; McHaourab, Lietzow, Hideg, & Hubbell, 1996). For α-helical membrane proteins, the spin label should be placed on the detergent-exposed helical sites, which will have an ordered backbone, but will likely not disrupt the structure, though there are many instances of the spin label introduced at tertiary contact sites in α-helical membrane proteins without disrupting fold or function (Claxton et al., 2010; Columbus et al., 2009; Gross, Columbus, Hideg, Altenbach, & Hubbell, 1999; Lo, Kroncke, Solomon, & Columbus, 2014; Perozo, Kloda, Cortes, & Martinac, 2001; Wegener, Tebbe, Steinhoff, & Jung, 2000). The CW EPR lineshapes at detergent-exposed α-helical sites are distinct and are slightly less mobile compared to their soluble protein counterparts (Columbus & Hubbell, 2002; Kroncke et al., 2010). Crystal structures of two spin-labeled sites on detergent-exposed sites of LeuT reveal that the spin label interacts with the surface of the membrane protein and restricts the motion of the nitroxide label (Kroncke et al., 2010). In the soluble protein counterparts, density for the spin label is often not observed due to a lack of interaction with the protein and oscillations about the two terminal bonds of the spin label (Guo, Cascio, Hideg, & Hubbell, 2008; Langen, Oh, Cascio, & Hubbell, 2000).
1.2 Choice of spin label The most well-studied spin label is MTSSL (Fig. 2A; referred to as R1 once incorporated into a protein). The internal dynamics, dihedral preferences,
334
Linda Columbus and Brett Kroncke
A
B Cβ
C Cβ
S
S
S
S
Cβ S S
N N
N O
N O
O
O
Figure 2 Structures of spin labels. The most common spin labels for PRE measurements MTSSL and the diamagnetic equivalent are shown in (A) and (B), respectively. An example of an alternative label with restricted motion is shown in (C).
and structure on helical sites of soluble and membrane proteins were determined and, thus, information crucial to the interpretation of PRE measurements is known for this spin label. However, there are some drawbacks in using this label: (i) the disulfide formed between the protein and label is labile resulting in increased “free” spin label over time, (ii) a contaminant is often observed in protein–detergent complexes and needs to be recognized and removed (Kroncke & Columbus, 2012), and (iii) oscillations about the internal side-chain bonds can contribute to a broad spatial distribution. To address the spatial distribution of the label, there are additional spin labels that have restricted motion compared to R1 (such as that shown in Fig. 2C) (Fawzi et al., 2011; Hubbell, Lopez, Altenbach, & Yang, 2013); however, the labels are larger, have not been investigated on membrane proteins, and may increase the nonspecifically bound contaminants observed in protein–detergent complexes (described below; Kroncke & Columbus, observation).
1.3 Protein topology and PRE restraint coverage In addition to the dynamic considerations of placement of the spin label, the PRE restraint coverage (the number and topological impact of each PRE restraint) required for an accurate structure calculation should be considered. Two research groups have investigated the influence of topological position, as well as the number of spin-labeled sites, required in order to obtain an
Solution NMR Structure Determination
335
accurate overall topology (Chen et al., 2011) and structure (Gottstein, Reckel, Dotsch, & Guntert, 2012). For overall topology, Chen et al. suggest that for α-helical membrane proteins with five to seven transmembrane helices two to three labels are sufficient. This minimal set of long-range restraints when combined with a sufficient number of RDC restraints and mediumrange intraresidue NOEs can lead to a well-determined solution NMR structure. Gottstein et al. determined that one spin label per helix was the minimum number of restraints to obtain correct structures. The spin label is best incorporated into the center of the helix (Gottstein et al., 2012) and facing the detergent (Chen et al., 2011). If labeling is only efficient toward the termini of the helices, then you will need to have two PRE restraint sets per helix. The main differences between these two studies are resolution and assumptions utilized in the structure calculation. Chen et al. were developing computational methods for using a minimal set of PREs to determine topology and Gottstein et al. aimed to evaluate PREs as sufficient restraints for high-resolution structure determination. Therefore, in the context of this review, a minimum of one label per helix is recommended.
1.4 Structure and function perturbation The R1 spin label is well tolerated in many regions of α-helical membrane proteins (Claxton et al., 2010; Gross et al., 1999; Lo et al., 2014; Perozo et al., 2001; Wegener et al., 2000). However, all spin-labeled mutants should be evaluated for disruption of fold and function. A major challenge is that functional assays are often hindered in detergent systems. The fold is best analyzed by recording the Heteronuclear Single Quantum Coherence (HSQC) spectrum of the protein labeled with the diamagnetically equivalent spin label (Fig. 2B) (Eichmann et al., 2014; Lo et al., 2014; Zhou et al., 2008); this spectrum needs to be recorded for the PRE measurement regardless and should also be evaluated for the probe’s influence on the global fold.
1.5 Labeling efficiency The labeling efficiency varies between sites on the protein and to some extent correlates with the accessibility of the reactive sulfhydryl. Typically, excess MTSSL is needed to drive the reaction to completion. Even at a molar ratio of 1:6 (protein:spin label), labeling overnight, the labeling efficiency is highly variable between different sites, with more occluded sites labeling less efficiently than exposed sites (Kelly & Gross, 2003;
336
Linda Columbus and Brett Kroncke
Kroncke & Columbus, 2012). The high labeling efficiency required for PRE measurements cannot be achieved unless high concentrations (relative to protein) of spin label are used.
1.6 Spin label contaminant In micellar systems, unreacted, nonspecifically associated spin label contaminant is prevalent and complicates the interpretation of relaxation data. To circumvent this challenge, a strategy of separation that includes affinity chromatography and incubation times is proposed by Kroncke & Columbus (2012). The contaminant is easily identified by a characteristic Electron Paramagnetic Resonance (EPR) lineshape and the EPR spectrum can be used to easily quantify the amount of contaminant (Kroncke & Columbus, 2012).
2. PRE MEASUREMENTS The nitroxide spin label enhances both longitudinal (R1) and transverse (R2) relaxation rates, and both measurements have been used for calculating PRE restraints for soluble proteins; however, only R2 measurements have been utilized for polytopic α-helical membrane proteins. In terms of nitroxide-derived PREs, R2 measurements are preferred because they are less sensitive to the internal motions of the nitroxide side chain (Iwahara, Schwieters, & Clore, 2004). For the PRE measurement, there are specific considerations at the acquisition and processing steps.
2.1 The reference spectrum Two TROSY-based 15N-HSQC spectra are acquired—one with the nitroxide present and the other with either the reduced nitroxide or the diamagnetically equivalent spin label. Ascorbic acid can be used to reduce the nitroxide; however, a nitroxide buried in a micelle is often not fully reduced with this method (Liang, Bushweller, & Tamm, 2006; Lo et al., 2014). The partial reduction could lead to an underestimate of distances and introduces variability between samples because of the dependence on the accessibility of the label to the reductant. A suggested alternative is the use of the diamagnetic spin label (Fig. 2B), although this approach doubles the number of samples needed. For the eight proteins listed in Table 1, two used the diamagnetic label (YgaP and DsbB), five used ascorbic acid (DAGK, ArcB, KdpD, QseC, and UCP2), and one used the unlabeled native protein as the reference spectrum (proteorhodopsin).
337
Solution NMR Structure Determination
2.2 Calculation of distances from intensity ratios Once the paramagnetic and diamagnetic spectra are acquired, Lorentzian peak models, which are fit to both dimensions, and intensity ratios (Ipara/Idia) are calculated for each assigned resonance (Battiste & Wagner, 2000). The intensities should be normalized using the average of a set of unperturbed resonance peaks (6 or more) to account for differences in protein concentrations. For resonance peaks that broadened beyond detection in the paramagnetic spectrum, the intensity can be estimated from the noise (Battiste & Wagner, 2000). Using the intensity ratio, the relaxation rate enhancements (R2sp) can be quantified by the following relationship: Ipara R2 eR2 t ¼ Idia R2 + R2sp sp
(1)
where R2 is the intrinsic relaxation rate for each amide and is estimated from the half-height Lorentzian linewidths in the diamagnetic spectrum, t is the total INEPT evolution time (9–12 ms), Ipara is the resonance peak intensity in the paramagnetic spectrum, and Idia is the resonance peak intensity of the diamagnetic spectrum (Battiste & Wagner, 2000; Gillespie & Shortle, 1997a, 1997b). Solving for R2sp can be done numerically or the exponential can be well approximated with the first-order term in a sp Taylor series expansion (i.e., eR2 t 1 R2sp t) resulting in the following relationship: Ipara R2 1 Idia R2sp : (2) Ipara + R2t Idia The modified Solomon–Bloembergen equation (Krugh, 1976; Solomon & Bloembergen, 1956) for transverse relaxation can be used to convert the relaxation enhancement rates into distances:
1=6 K 3τc 4τc + r¼ 1 + ω2h τ2c R2sp
(3)
where r is the electron–nuclear distance, ωh is the Larmor frequency of the nuclear spin, τc is the correlation time for the electron–nuclear interaction and is estimated from the global protein correlation time from 15N relaxation
338
Linda Columbus and Brett Kroncke
measurements, and K ¼ 151 SðS + 1Þγ 2 g2 β2 ¼ 1:23 1032 cm6 s2 , where g is the electron g-factor, γ is the nuclear gyromagnetic ratio, and β is the Bohr magneton.
2.3 Distance classifications The distance restraints are typically classified into three categories based on the intensity ratio (Battiste & Wagner, 2000; Liang et al., 2006), although the intensity ratio cutoff values and bounds have varied in different structure calculations. Resonances that are not significantly broadened (Ipara/Idia > 0.85) can be treated as unrestrained or only restrained with a lower bound distance calculated for the 0.85 intensity ratio. For resonances with intensity ratios between 0.15 and 0.85, the intensity ratio versus distance is relatively linear; thus, the calculated distance from Eq. (3) is used. For peaks broadened beyond detection or with an intensity ratio
Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins: A Guide to Spin Label Paramagnetic Relaxation Enhancement Restraints Linda Columbus*,1, Brett Kroncke† *Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA † Department of Biochemistry and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Site-Directed Spin Labeling 1.1 Nitroxide dynamics 1.2 Choice of spin label 1.3 Protein topology and PRE restraint coverage 1.4 Structure and function perturbation 1.5 Labeling efficiency 1.6 Spin label contaminant 2. PRE Measurements 2.1 The reference spectrum 2.2 Calculation of distances from intensity ratios 2.3 Distance classifications 3. Structure Calculation with PRE Restraints 3.1 The nitroxide spin label 3.2 Error bounds for PRE distances 3.3 Planar bilayer restraints 4. Assessment of Structure Quality 5. Future Developments 5.1 Molecular dynamics, explicit solvent, and the native bilayer 5.2 Carbon PREs 6. Summary References
Methods in Enzymology, Volume 557 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.12.005
#
2015 Elsevier Inc. All rights reserved.
332 333 333 334 335 335 336 336 336 337 338 338 338 341 341 341 342 342 343 343 343
329
330
Linda Columbus and Brett Kroncke
Abstract Solution nuclear magnetic resonance structures of polytopic α-helical membrane proteins require additional restraints beyond the traditional Nuclear Overhauser Effect (NOE) restraints. Several methods have been developed and this review focuses on paramagnetic relaxation enhancement (PRE). Important aspects of spin labeling, PRE measurements, structure calculations, and structural quality are discussed.
Polytopic α-helical membrane proteins are challenging systems for solution nuclear magnetic resonance (NMR) approaches. Four major bottlenecks are (i) membrane protein expression in sufficient yields required for in vitro investigations; (ii) extraction, solubilization, and stabilization of folded and functional membrane proteins in membrane mimics such as detergents; (iii) structural heterogeneity and dynamics that are integral to membrane protein function, but interfere with in vitro structural investigations; and (iv) Nuclear Overhauser Effect restraints (NOEs)—the workhorse of NMR structure calculations—do not provide tertiary structural information for deuterated α-helical membrane proteins. For the most recent reviews of challenges i–iii, readers should seek these references for expression (Guerfal et al., 2013; Gupta, Kueppers, & Schmitt, 2014; Kimura-Soyema, Shirouzu, & Yokoyama, 2014; Parker & Newstead, 2014; Su, Si, Baker, & Berger, 2013), solubilization (Columbus et al., 2009; Duquesne & Sturgis, 2010; Schimerlik, 2001), and structural heterogeneity (Cafiso, 2014; Pan, Piyadasa, O’Neil, & Konermann, 2012). There are several methods (e.g., residual dipolar couplings (RDCs) and methyl NOEs) developed to tackle the lack of NOE-based structural restraints and this review will focus on nitroxide spin label paramagnetic relaxation enhancement (PRE)-derived restraints as applied to polytopic α-helical membrane proteins. PRE measurements rely on the incorporation of a paramagnetic species site specifically (e.g., a nitroxide spin label), which enhances the relaxation of neighboring NMR active nuclei in a distance-dependent manner. PRE-proton-derived ˚ ) compared to the standard 1H–1H NOE distances are long range (15–25 A (5–6 A˚) and were used in the solution NMR structure determination of eight (Berardi, Shih, Harrison, & Chou, 2011; Eichmann et al., 2014; Maslennikov et al., 2010; Reckel et al., 2011; Van Horn et al., 2009; Zhou et al., 2008) of the nine polytopic α-helical membrane proteins determined to date (Table 1; with sensory rhodopsin the exception (Gautier & Nietlispach, 2012)). The general workflow for utilizing PRE restraints for the NMR structure determination of α-helical membrane proteins is shown in Fig. 1 and is divided into
Table 1 Solution NMR structures of polytopic integral membrane proteins Restraints in addition to backbone NOEsa PDB IDs
SL-PRE
RDCs
Inter-NOEsb
39
DsbB
2k73, 2k74 1144
337
DAGK
2kdc
67
208
Sensory rhodopsin 2ksy
1536
# of TMa Oligomeric state Year
X-ray structure rmsd (Å)
4
Monomer 2008 2zuq
2.4
3
Trimer
d
7
Monomer 2010 1h68
2009 3ze3
KdpD
2ksf
845
4
Monomer 2010 None
ArcB
2ksd
291
2
Monomer 2010 None
QseC
2kse
295
2
Monomer 2010 None
Proteorhodopsin
2l6x
1006
81
7
Monomer 2011 4jq6c
UCP2
2lck
452
470
6
Monomer 2011 None
YgaP
2mpn
8
2
Dimer
a
87
216
Per monomer. Nonsequential and long range (j1 jj 5). c Protein with 57% identity. d The NMR structure has domain swapping and the crystal structure does not. b
2014 None
1.06
3.71
332
Linda Columbus and Brett Kroncke
■
Introduce unique individual cysteine residues on each TM segment
Choose sites that are in ordered regions of the protein
■
SDSL
Assess the dynamics of the label using CW EPR lineshape analysis
Spin label each site with MTSSL and the diamagnetic equivalent
■
Measure labeling efficiency
■
Determine labels do not influence the global fold
Record the HSQC of each MTSSL-labeled site
PRE measurement
Record the HSQC of each diamagnetically labeled site
■
Normalize peak intensities to account for differences in protein concentration
Measure intensity ratios for each assigned resonance Bin the intensity ratios into three classes
Structure calculation
Combine PRE, RDC, methyl NOE, sequential NOE, hydrogen bonding restraints with chemical shift-derived dihedral angles
■
Model the nitroxide spin label at each position labeled and use the c1 – c3 dihedral restraints for the nitroxide side chain
Calculate ~300 initial structures
■
Use a ±4 Å error bounds on the PRE distance
Select the 20 lowest energy structures
Structure analysis
Comparison of calculated chemical shift from ensemble to observed chemical shifts
rmsd of ensemble
Analysis of helix packing: interhelical crossing angles, interhelical distances, and helical kinks
Figure 1 Workflow diagram for utilizing PRE restraints for the NMR structure determination of α-helical membrane proteins. There are four steps: (i) site-directed spin labeling (SDSL), (ii) the PRE measurement, (iii) the structure calculation, and (iv) the evaluation of the accuracy and precision of the structure. Tips are also provided in the text on the right.
four stages: (i) site-directed spin labeling (SDSL), (ii) PRE measurement, (iii) structure calculation, and (iv) accuracy and precision evaluation of the structure.
1. SITE-DIRECTED SPIN LABELING Typically, SDSL is used to introduce a nitroxide spin label at a unique cysteine residue. Mutagenesis introduces alanine or serine in place of native cysteine residues such that a single cysteine can be introduced throughout
Solution NMR Structure Determination
333
the α-helical membrane protein. Selection of where to place the cysteine residue, and subsequently the spin label, requires a few important considerations.
1.1 Nitroxide dynamics The dynamics of the spin label, which is determined by internal side-chain motions, backbone fluctuations, and rigid body motions, can contribute to large ranges of distances between the label and the relaxed nuclei. However, the PRE measurement is most influenced by the closer distances; the distance range interpreted may only capture a subpopulation of states sampled. Therefore, placing the spin label in dynamic regions may complicate the interpretation of the structure calculated and is best avoided by placing the spin label in structured regions. The dynamics of the spin label, which reflect both the internal flexibility of the spin label and the flexibility of the protein backbone, should always be evaluated using CW EPR (Columbus, Kalai, Jeko, Hideg, & Hubbell, 2001; Hubbell, McHaourab, Altenbach, & Lietzow, 1996; Kroncke, Horanyi, & Columbus, 2010; McHaourab, Lietzow, Hideg, & Hubbell, 1996). For α-helical membrane proteins, the spin label should be placed on the detergent-exposed helical sites, which will have an ordered backbone, but will likely not disrupt the structure, though there are many instances of the spin label introduced at tertiary contact sites in α-helical membrane proteins without disrupting fold or function (Claxton et al., 2010; Columbus et al., 2009; Gross, Columbus, Hideg, Altenbach, & Hubbell, 1999; Lo, Kroncke, Solomon, & Columbus, 2014; Perozo, Kloda, Cortes, & Martinac, 2001; Wegener, Tebbe, Steinhoff, & Jung, 2000). The CW EPR lineshapes at detergent-exposed α-helical sites are distinct and are slightly less mobile compared to their soluble protein counterparts (Columbus & Hubbell, 2002; Kroncke et al., 2010). Crystal structures of two spin-labeled sites on detergent-exposed sites of LeuT reveal that the spin label interacts with the surface of the membrane protein and restricts the motion of the nitroxide label (Kroncke et al., 2010). In the soluble protein counterparts, density for the spin label is often not observed due to a lack of interaction with the protein and oscillations about the two terminal bonds of the spin label (Guo, Cascio, Hideg, & Hubbell, 2008; Langen, Oh, Cascio, & Hubbell, 2000).
1.2 Choice of spin label The most well-studied spin label is MTSSL (Fig. 2A; referred to as R1 once incorporated into a protein). The internal dynamics, dihedral preferences,
334
Linda Columbus and Brett Kroncke
A
B Cβ
C Cβ
S
S
S
S
Cβ S S
N N
N O
N O
O
O
Figure 2 Structures of spin labels. The most common spin labels for PRE measurements MTSSL and the diamagnetic equivalent are shown in (A) and (B), respectively. An example of an alternative label with restricted motion is shown in (C).
and structure on helical sites of soluble and membrane proteins were determined and, thus, information crucial to the interpretation of PRE measurements is known for this spin label. However, there are some drawbacks in using this label: (i) the disulfide formed between the protein and label is labile resulting in increased “free” spin label over time, (ii) a contaminant is often observed in protein–detergent complexes and needs to be recognized and removed (Kroncke & Columbus, 2012), and (iii) oscillations about the internal side-chain bonds can contribute to a broad spatial distribution. To address the spatial distribution of the label, there are additional spin labels that have restricted motion compared to R1 (such as that shown in Fig. 2C) (Fawzi et al., 2011; Hubbell, Lopez, Altenbach, & Yang, 2013); however, the labels are larger, have not been investigated on membrane proteins, and may increase the nonspecifically bound contaminants observed in protein–detergent complexes (described below; Kroncke & Columbus, observation).
1.3 Protein topology and PRE restraint coverage In addition to the dynamic considerations of placement of the spin label, the PRE restraint coverage (the number and topological impact of each PRE restraint) required for an accurate structure calculation should be considered. Two research groups have investigated the influence of topological position, as well as the number of spin-labeled sites, required in order to obtain an
Solution NMR Structure Determination
335
accurate overall topology (Chen et al., 2011) and structure (Gottstein, Reckel, Dotsch, & Guntert, 2012). For overall topology, Chen et al. suggest that for α-helical membrane proteins with five to seven transmembrane helices two to three labels are sufficient. This minimal set of long-range restraints when combined with a sufficient number of RDC restraints and mediumrange intraresidue NOEs can lead to a well-determined solution NMR structure. Gottstein et al. determined that one spin label per helix was the minimum number of restraints to obtain correct structures. The spin label is best incorporated into the center of the helix (Gottstein et al., 2012) and facing the detergent (Chen et al., 2011). If labeling is only efficient toward the termini of the helices, then you will need to have two PRE restraint sets per helix. The main differences between these two studies are resolution and assumptions utilized in the structure calculation. Chen et al. were developing computational methods for using a minimal set of PREs to determine topology and Gottstein et al. aimed to evaluate PREs as sufficient restraints for high-resolution structure determination. Therefore, in the context of this review, a minimum of one label per helix is recommended.
1.4 Structure and function perturbation The R1 spin label is well tolerated in many regions of α-helical membrane proteins (Claxton et al., 2010; Gross et al., 1999; Lo et al., 2014; Perozo et al., 2001; Wegener et al., 2000). However, all spin-labeled mutants should be evaluated for disruption of fold and function. A major challenge is that functional assays are often hindered in detergent systems. The fold is best analyzed by recording the Heteronuclear Single Quantum Coherence (HSQC) spectrum of the protein labeled with the diamagnetically equivalent spin label (Fig. 2B) (Eichmann et al., 2014; Lo et al., 2014; Zhou et al., 2008); this spectrum needs to be recorded for the PRE measurement regardless and should also be evaluated for the probe’s influence on the global fold.
1.5 Labeling efficiency The labeling efficiency varies between sites on the protein and to some extent correlates with the accessibility of the reactive sulfhydryl. Typically, excess MTSSL is needed to drive the reaction to completion. Even at a molar ratio of 1:6 (protein:spin label), labeling overnight, the labeling efficiency is highly variable between different sites, with more occluded sites labeling less efficiently than exposed sites (Kelly & Gross, 2003;
336
Linda Columbus and Brett Kroncke
Kroncke & Columbus, 2012). The high labeling efficiency required for PRE measurements cannot be achieved unless high concentrations (relative to protein) of spin label are used.
1.6 Spin label contaminant In micellar systems, unreacted, nonspecifically associated spin label contaminant is prevalent and complicates the interpretation of relaxation data. To circumvent this challenge, a strategy of separation that includes affinity chromatography and incubation times is proposed by Kroncke & Columbus (2012). The contaminant is easily identified by a characteristic Electron Paramagnetic Resonance (EPR) lineshape and the EPR spectrum can be used to easily quantify the amount of contaminant (Kroncke & Columbus, 2012).
2. PRE MEASUREMENTS The nitroxide spin label enhances both longitudinal (R1) and transverse (R2) relaxation rates, and both measurements have been used for calculating PRE restraints for soluble proteins; however, only R2 measurements have been utilized for polytopic α-helical membrane proteins. In terms of nitroxide-derived PREs, R2 measurements are preferred because they are less sensitive to the internal motions of the nitroxide side chain (Iwahara, Schwieters, & Clore, 2004). For the PRE measurement, there are specific considerations at the acquisition and processing steps.
2.1 The reference spectrum Two TROSY-based 15N-HSQC spectra are acquired—one with the nitroxide present and the other with either the reduced nitroxide or the diamagnetically equivalent spin label. Ascorbic acid can be used to reduce the nitroxide; however, a nitroxide buried in a micelle is often not fully reduced with this method (Liang, Bushweller, & Tamm, 2006; Lo et al., 2014). The partial reduction could lead to an underestimate of distances and introduces variability between samples because of the dependence on the accessibility of the label to the reductant. A suggested alternative is the use of the diamagnetic spin label (Fig. 2B), although this approach doubles the number of samples needed. For the eight proteins listed in Table 1, two used the diamagnetic label (YgaP and DsbB), five used ascorbic acid (DAGK, ArcB, KdpD, QseC, and UCP2), and one used the unlabeled native protein as the reference spectrum (proteorhodopsin).
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2.2 Calculation of distances from intensity ratios Once the paramagnetic and diamagnetic spectra are acquired, Lorentzian peak models, which are fit to both dimensions, and intensity ratios (Ipara/Idia) are calculated for each assigned resonance (Battiste & Wagner, 2000). The intensities should be normalized using the average of a set of unperturbed resonance peaks (6 or more) to account for differences in protein concentrations. For resonance peaks that broadened beyond detection in the paramagnetic spectrum, the intensity can be estimated from the noise (Battiste & Wagner, 2000). Using the intensity ratio, the relaxation rate enhancements (R2sp) can be quantified by the following relationship: Ipara R2 eR2 t ¼ Idia R2 + R2sp sp
(1)
where R2 is the intrinsic relaxation rate for each amide and is estimated from the half-height Lorentzian linewidths in the diamagnetic spectrum, t is the total INEPT evolution time (9–12 ms), Ipara is the resonance peak intensity in the paramagnetic spectrum, and Idia is the resonance peak intensity of the diamagnetic spectrum (Battiste & Wagner, 2000; Gillespie & Shortle, 1997a, 1997b). Solving for R2sp can be done numerically or the exponential can be well approximated with the first-order term in a sp Taylor series expansion (i.e., eR2 t 1 R2sp t) resulting in the following relationship: Ipara R2 1 Idia R2sp : (2) Ipara + R2t Idia The modified Solomon–Bloembergen equation (Krugh, 1976; Solomon & Bloembergen, 1956) for transverse relaxation can be used to convert the relaxation enhancement rates into distances:
1=6 K 3τc 4τc + r¼ 1 + ω2h τ2c R2sp
(3)
where r is the electron–nuclear distance, ωh is the Larmor frequency of the nuclear spin, τc is the correlation time for the electron–nuclear interaction and is estimated from the global protein correlation time from 15N relaxation
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measurements, and K ¼ 151 SðS + 1Þγ 2 g2 β2 ¼ 1:23 1032 cm6 s2 , where g is the electron g-factor, γ is the nuclear gyromagnetic ratio, and β is the Bohr magneton.
2.3 Distance classifications The distance restraints are typically classified into three categories based on the intensity ratio (Battiste & Wagner, 2000; Liang et al., 2006), although the intensity ratio cutoff values and bounds have varied in different structure calculations. Resonances that are not significantly broadened (Ipara/Idia > 0.85) can be treated as unrestrained or only restrained with a lower bound distance calculated for the 0.85 intensity ratio. For resonances with intensity ratios between 0.15 and 0.85, the intensity ratio versus distance is relatively linear; thus, the calculated distance from Eq. (3) is used. For peaks broadened beyond detection or with an intensity ratio
