. Author manuscript; available in PMC: 2009 Feb 4.

Published in final edited form as:Biochim Biophys Acta. 2007 Dec 14;1778(4):937–944. doi:10.1016/j.bbamem.2007.11.013

Solution NMR of signal peptidase, a membrane protein

Monika Musial-Siwek¹,Debra A Kendall¹,Philip L Yeagle^1,^*

¹Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269, USA

Corresponding author. Department of Molecular and Cell Biology, 91 North Eagleville Road, The University of Connecticut, Storrs, CT 06269, USA. Tel.: +1 860 486 5154.E-mail address:yeagle@uconn.edu (P.L.Yeagle).

Issue date 2008 Apr.

PMC Copyright notice

PMCID: PMC2635912 NIHMSID: NIHMS83570 PMID:18177734

Abstract

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly²H,¹³C,¹⁵N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.

Keywords: Signal peptidase, NMR, Membrane protein

1. Introduction

Extensive structural data on soluble proteins are available, but reports on structures of integral membrane proteins with multiple membrane spanning segments still represent less than 1/2% of the database of protein structures [1,2]. Membrane proteins carry out critical physiological functions such as signaling, protein secretion and ion transport, and thus are key targets for drug development. Elucidating the structures of membrane proteins will aid our understanding of their activity, ligand binding sites, and potential interaction with therapeutic agents.

Many difficulties arise when preparing a membrane protein for structural analysis. First, it is challenging to obtain high-yield expression and purification of membrane proteins. A second challenge for membrane protein preparation is the requirement for lipid mimetic environments, such as detergents, to keep these hydrophobic proteins in a native conformation after extraction from their natural environment, the lipid bilayer. Third, it is exceptionally difficult to analyze membrane protein structure with conventional methods such as electron microscopy, X-ray crystallography or NMR spectroscopy. Electron microscopy usually provides only a low-resolution structure of the membrane protein and it is difficult to obtain high quality crystals of proteins from detergent micelles for X-ray analysis. Difficulties with the NMR experiments arise from the large size of many membrane proteins and this leads to relatively slow rotational correlation times and consequent resonance broadening. The overall size of the complex increases when the protein is associated with detergent micelles exacerbating this problem.

NMR spectroscopy on high molecular weight systems has advanced significantly with the introduction of TROSY experiments [3]. TROSY has enabled the NMR analysis of larger proteins than was previously possible. Using this NMR methodology several membrane protein structures have been investigated (for review see [4]). A number of structures of β-barrel proteins from bacterial outer membranes (up to 20 kD) have been analyzed using NMR techniques [5-7]. Structures of integral membrane proteins solubilized in organic solvents have also emerged [8,9], although organic solvents might be unsuitable for proteins with large extramembraneous segments [2]. Using NMR methods, some structural data have been obtained from integral membrane protein structures containing multiple helical transmembrane segments [10-14]. However, only one complete chemical shift assignment of an intact integral membrane protein with a transmembrane helical bundle in detergent micelles has been reported thus far [15], and no complete three-dimensional structures have yet been reported in the literature.

Here we useEscherichia coli signal peptidase I (SPase I) as a model membrane-bound protein for the development of methods for NMR structural determination. SPase I is found in the membranes of Gram-negative and Gram-positive bacteria, the endoplasmic reticulum (ER), chloroplast, and mitochondria (for reviews see [16-18]). The enzyme cleaves the amino-terminal signal peptide from a secreted protein during the final stages of membrane translocation and consequently, plays a critical role in protein targeting.

SPase I is a protein with two transmembrane segments (residues 4-28 and 58-76), a cytoplasmic loop (residues 29-57), and a periplasmic C-terminal catalytic domain (residues 77-323). Although a crystal structure of the soluble C-terminal catalytic domain of the SPase I has been reported [19-21], there is no structural information on the full-length protein. Therefore, the structural relationship between the catalytic domain and the transmembrane domain is unknown.

Here we report solution NMR data from full-length SPase I, including the transmembrane domain. We expressed, isotopically labeled, and purified the protein in quantity sufficient for NMR analysis at 600 MHz and 900 MHz. The SPase I was active under the conditions of the NMR experiment. Data from uniformly²H,¹³C,¹⁵N-labeled intact SPase I show interpretable¹H-¹⁵N TROSY-HSQC, HNCACB, HNCA and HN (CO) NMR spectra. The most readily visible resonances arise from the large C-terminal domain of the protein.

2. Materials and methods

2.1. Materials

Deuterium oxide (99.9%), ammonium chloride (>99%¹⁵N-labeled), and glucose (uniformly >N99%¹³C-labeled) were purchased from Spectra Stable Isotopes (Columbia, MD). Dnase I (Amplification Grade) was obtained from Invitrogen Corp. (Carlsbad, CA),n-octyl-β-D-glucopyranoside (OG) (Calbiochem, La Jolla, CA), dodecylphosphocholine (DPC) (Avanti, Polar Lipids, Inc., Alabaster, AL), Q-Sepharose (GE Healthcare, Piscataway, NJ), and Ni-agarose were from Qiagen (Valencia, CA).

2.2. Signal peptidase expression and purification

BL21(DE3) cells transfected with the pET23b plasmid (generously provided by Ross Dalbey, The Ohio State University), coding forE. coli 6-His-tagged SPase I, were grown in M9 Minimal Media labeled with²H,¹³C and¹⁵N (²H,¹³C,¹⁵N-M9 media): 99.9% D₂O, 11.1 mM¹³C-labeled glucose, and 18.7 mM¹⁵N-labeled ammonium chloride. First, the cells were grown in 25 ml of 50%/50%²H/¹H,¹³C,¹⁵N-M9 media overnight to reach OD=0.7, then 20 ml of cells were spun for 10 min at 13,000 rpm sterilely and resuspended in 50 ml²H,¹³C,¹⁵N-M9 media. The cells were grown to reach OD₆₀₀=0.84, spun again sterilely for 10 min at 13,000 rpm and resuspended in 500 ml²H,¹³C,¹⁵N-M9 media. Starting with OD₆₀₀=0.18 the cells were grown at 37 °C with shaking at 230 rpm until OD₆₀₀=0.5 followed by induction with 0.5 mM IPTG for 4 h. Cells were harvested by centrifugation at 6000 rpm for 20 min, and resuspended in 25 ml of lysis buffer (50 mM Tris, pH 8.0, 20% glucose). The solution was mixed, after addition of lysozyme (6 mg) and Dnase I, for 10 min at room temperature and frozen at -70 °C overnight. The labeled SPase I was purified as described previously by Klenotic et al. [22] with some modifications. Cells were thawed and 200 μl of 1 M magnesium acetate was added to the sample. The sample was mixed, centrifuged, and the pellet resuspended in 10 mM triethanolamine, 10% glycerol, pH 7.9. Following centrifugation, the pellet was homogenized in solubilization buffer (10 mM triethanolamine, 10% glycerol, 1% Triton X-100, pH 7.9), centrifuged again and the supernatant loaded on a Q-Sepharose column. The SPase I was eluted with a continuous gradient of 0-0.1 M KCl. Fractions containing SPase I were loaded onto Ni-agarose resin equilibrated in 6-His buffer (10 mM Tris, pH 8.5, 100 mM KCl, 20 mM imidazole, 10 mM B-mercaptoethanol, and 1% Triton X-100) and eluted with a step gradient of imidazole (100-300 mM). To remove the imidazole and prepare the sample for NMR spectroscopy, the protein was concentrated and buffer exchanged with 20 mM phosphate buffer pH 6.3, 10 mM EDTA (added to negate possible Ni interference) and 0.3% Triton X-100 using a Centricon-10 membrane (Millipore, Billerica, MA). Protein concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford, IL).

2.3. Detergent exchange

To exchange detergents, the Ni-agarose resin bound with SPase I was washed 3 times with 1% of one of the three detergents, Triton X-100, OG, or DPC in 6-His buffer and spun at very low speed (1000 rpm) to separate the resin-bound protein from the buffer. SPase I was eluted with four washes of 300 mM imidazole. To remove the imidazole and prepare sample for the NMR, the protein was concentrated and buffer exchanged with 20 mM NaH₂PO₄ pH 6.3, 10 mM EDTA and the appropriate detergent using a Centricon-10 membrane. SPase I in each detergent was incubated at 22 °C and 37 °C for one week and checked for stability by monitoring for degradation products on SDS-PAGE gels and by testing the activity (see below).

2.4. Activity assay

GST fused to the N-terminus of the alkaline phosphatase signal peptide (SP) and first 30 amino acids of the mature protein (AP), and 6-His-tagged (His) at the C-terminus (GST-SP-AP-His), was used as a substrate for SPase I proteolysis [23]. Signal peptidase was incubated at different concentrations with GST-SP-AP-His in Tris buffer pH 8, and 1% Triton X-100, or 1% OG, or 1% DPC at 37 °C for 2 h. The reaction was stopped by addition of sample buffer. The sample was run on SDS-PAGE and silver stained.

2.5. NMR experiments

All experiments were performed on either a 900 MHz Bruker or a 600 MHz Varian spectrometer at 22 °C. 2D¹H-¹⁵N TROSY-HSQC, 3D HNCACB, 3D HNCA and 3D HN(CO) spectra were collected on²H,¹³C,¹⁵N-labeled SPase I at 12 mg/ml in 20 mM NaH₂PO₄, 10 mM EDTA, pH 6.3 and 1% DPC. This detergent concentration was well above the critical micelle concentration for DPC and from the molar ratio (DPC/SPase I is about 93) it can be estimated that about 1 protein molecule, on average, was in each detergent micelle. The¹H-¹⁵N HSQC spectra were acquired as 2048 and 128 complex points in¹H and¹⁵N dimension, respectively. For the SPase I sequence specific assignments, a combination of 3D HNCACB, 3D HNCA and 3D HN(CO) spectra was utilized. 3D HNCACB data were acquired with 1024 points in the¹H dimension, 64 points in the¹⁵N dimension, and 32 points in the¹³C dimension, while being averaged over 8 transients. 3D HNCA were acquired with 1024 points in the¹H dimension, 64 points in the¹⁵N dimension, and 16 points in the¹³C dimension over 16 transients. 3D HN(CO) data were acquired with 1024 points in the¹H dimension, 64 points in the¹⁵N dimension, and 16 points in the¹³C dimension over 16 transients. All spectra were processed using nmrPipe/nmrDraw software [24] and analyzed using the program Sparky (http://www.cgl.ucsf.edu/home/sparky).

3. Results

3.1. Isolation of stable-isotope labeled SPase I

Since NMR is a relatively insensitive technique, an expression system that provides a high yield of membrane protein labeled with stable isotopes is critical. Therefore, the production of SPase I from BL21 (DE3) cells transfected with the pET23b-coding SPase I plasmid, in LB media, then in minimal media, was optimized. Subsequently, the expression of SPase I was examined in minimal media in the presence of deuterium oxide (D₂O). No significant difference in expression levels was observed in the presence and absence of D₂O, although the cells grew much more slowly in D₂O. For example, to reach OD₆₀₀ of 0.5 the growth time increased by 40% in minimal media and 70% in minimal media containing D₂O, compared to the cell growth in LB media. We found that adaptation of the cells to the D₂O containing media was unnecessary. We also explored different induction times and found that a point, intermediate in log phase, was critical in maximizing expression level and minimizing degradation of the expressed protein. In addition, there was no difference observed in SPase I expression in the presence of different percentages of D₂O, or when MOPS media was used instead of M9 media. It proved possible to obtain 8.75 mg (12 mg/ml) of protein from 2 L of²H,¹³C,¹⁵N-labeled M9 media at about 95% purity (Fig. 1A).

Fig. 1 — Analysis ofE. coli SPase I stability and activity under different conditions. (A) Silver-stained SDS-PAGE of purified SPase I indicating the enzyme is about 95% pure. (B) Analysis of SPase I activity on silver stained SDS-PAGE. Different concentrations, as indicated, of the substrate (GST-SP-AP-His) were incubated with 1 μM SPase I over 2 h at 37 °C. The location of SPase I, GST-SP-AP-His, and the cleavage product, GST-SP, is indicated on the gel. (C) Comparison of the degradation of SPase I in Triton X-100, OG, and DPC detergent micelles via silver stained SDS-PAGE. Incubation time and temperature are indicated. The band corresponding to SPase I is marked and two of the degradation products are indicated by ● and ○. While, some discoloring is present on the scanned image in the last lane, the bands still can be distinguished. (D) Analysis of SPase I activity conducted as in (B). SPase I was incubated over 2 days in DPC micelles at different temperatures, as indicated, prior to the activity analysis. The band corresponding to SPase I is marked and two of the degradation products are indicated by ● and ○. (E) Analysis of SPase I activity conducted as in (B). SPase I was incubated over 7 days in DPC micelles at different temperatures, as indicated, prior to the activity analysis. The band corresponding to SPase I is marked and two of the degradation products are indicated by ● and ○.

3.2. Activity of purified, stable-isotope labeled SPase I

The activity of the stable-isotope labeled SPase I was monitored using an established assay [22,25]. In this assay, SPase I recognizes the signal peptide of the GST-SP-AP-His construct and cleaves the assembly into two fragments (C-terminal to the -1 position of the signal peptide). The two fragments are: (1) the GST protein plus the signal peptide (28 kD), and (2) the first 30 amino acids of the mature region 6-His-tagged (4 kD). On the gel, the appearance of the GST-SP band (28 kD) becomes evident, while the intensity of the band corresponding to the GST-SP-AP-His (32 kD) decreases in the presence of the enzyme relative to without the enzyme (Fig. 1B). These observations indicate that the GST-SP-AP-His fusion protein is cleaved by SPase I and reveal that the enzyme activity of the expressed protein is comparable to previously published assays [22,25]. Interestingly, the enzyme is more rapidly proteolyzed in the presence of the substrate, which would indicate that the GST-SP-AP-His activates the enzyme to some extent.

3.3. Detergent and temperature effects on protein stability

Because NMR experiments require long collection times, it is critical to find conditions under which the protein is stable for the duration of NMR data collection. We investigated three detergents (Triton X-100, OG and DPC) to determine which type of detergent micelles are better suited to maintain the stability of SPase I. Triton X-100 is a detergent often used to solubilize membrane proteins and SPase I is active in this detergent [26]. However, SPase I is unstable in Triton X-100 when incubated for a long time at elevated temperature. For example, after incubating the sample at 30 °C over 24 h white precipitate could be seen. Consistent with our observations, Wang et al. [27] established that SPase I is autolysed to a greater extent in Triton X-100 than in lipid vesicles (50% PC:50% PE).

The extent of SPase I degradation and its activity was tested in the presence of other detergent micelles. Another detergent previously used successfully to purify the enzyme was 1% OG [22]. Therefore this detergent (1%) and a detergent commonly used for NMR experiments with membrane proteins, 1% DPC [15], were both tested for their ability to maintain stable SPase I and were compared with 1% Triton X-100. FromFig. 1C it is apparent that SPase I is degraded in the presence of all three detergents, Triton X-100, OG and DPC, over time (e.g. compare 1 and 7 day incubations at 22 °C). The slowest degradation occurred in DPC. Elevated temperature enhances enzyme degradation: at 37 °C, the protein is autolysed and degraded more than at 22 °C (e.g. a lower molecular weight degradation product becomes evident). At 37 °C, degradation occurred to a lesser extent in DPC micelles than in Triton X-100 or OG. It has been reported that the autolysis site is located between two helices, at Ala40 and Ala41, consistent with our observation, generating a 31.5-kD size fragment after the cleavage [27]. SPase I activity was comparable in all three detergents (data not shown). Therefore, the following experiments were carried out with SPase I in DPC detergent micelles.

The temperature-dependent degradation and loss of SPase I activity in DPC micelles was determined. Temperature is important for the NMR experiment. Large complexes tumble very slowly leading to broadening of the NMR signal. The rotational correlation time of the protein in solution decreases with increase in temperature. Therefore, increasing the temperature would be expected to narrow resonances, resulting in a better-resolved NMR spectrum. However, the disadvantage of higher temperature is that SPase I is activated at higher temperature escalating the autolysis and leading to loss of enzyme activity. Consequently, we analyzed the enzyme activity after incubation as a function of time at 22 °C and 37 °C. InFig. 1D and E, the predominant band at the molecular weight corresponding to SPase I was evident. Autolysed SPase I (marked with a closed circle and co-migrating with the substrate) and bands at lower molecular weight (marked with an open circle), indicating degradation of the protein, appeared after two days and became more predominant after seven days of incubation. These signs of degradation were more prominent at 37 °C with autolysed SPase I further degrading to smaller fragments. After seven days of incubation, some fraction of the enzyme remained active at either temperature though less full-length enzyme was available at 37 °C. To ensure that the quality of the sample did not vary significantly in different experiments collected at different times following the purification of the protein, the temperature used to collect the NMR data was 22 °C.

3.4. HSQC spectrum of signal peptidase

High-resolution NMR spectra were obtained from intact SPase I in DPC detergent micelles.Fig. 2 shows the 600 MHz¹H-¹⁵N TROSY-HSQC spectrum of uniformly²H,¹³C,¹⁵N-labeled SPase I in DPC micelles. (The¹H-¹⁵N TROSY-HSQC spectrum acquired on the 900 MHz spectrometer did not differ substantially from the spectrum acquired on the 600 MHz instrument (data not shown).) In the 2D¹H-¹⁵N HSQC, many modestly resolved peaks were observed. Among these, approximately 24 Gly peaks were observed in the area of the HSQC spectrum where Gly residues were expected. In the SPase I amino acid sequence there are 28 Gly residues and of these, 4 Gly residues are located in the transmembrane segments.

The sample was also examined with magic angle spinning (MAS) NMR spectroscopy. MAS NMR has been used extensively to study membrane proteins in membrane bilayers [28-32]. Considering the size of SPase I in association with detergent micelles and the fact that large complexes do not tumble rapidly in solution, we combined solution NMR with MAS NMR to determine whether high resolution MAS (HRMAS) would increase resolution of the resonances. Comparison of HRMAS¹H,¹⁵N 2D-HSQC (on a 500 MHz spectrometer) with static¹H,¹⁵N 2D-HSQC (on a 600 MHz spectrometer), revealed no detectable improvement in resolution (data not shown).

3.5. Sequential resonance assignment

Collection and analysis of 3D HNCACB, 3D HNCA and 3D HN(CO) experiments allowed us to sequentially assign segments of the protein amino acid sequence using²H,¹³C,¹⁵N-labeled samples. InFig. 3, selected¹⁵N planes of sequential NMR spin system connectivities from a 600 MHz 3D HNCACB spectrum of uniformly²H,¹³C,¹⁵N-labeled SPase I are shown. From these data it was possible to sequentially assign sets of contiguous residues as follow: 98I-99G, 109Y-110G, 200R-202G, 205A-208G, 215N-217T, 221G-224L, 272M-275R, 278N-282S, and 308E-309G (Table 1).

Fig. 3 — Sequential assignment of SPase I residues. Some of the strip plots acquired from the HNCACB experiment are shown with residues/numbers indicated above the plots and chemical shifts shown at the bottom of the plot for¹H and¹⁵N and on the side for¹³C. The C^α (orange) and C^β (blue) have opposite signs. Correlated peaks are connected with lines.

Table 1.

Sequential resonance assignment of uniformly²H,¹³C,¹⁵N-labeled SPase I amino acid sequence from chemical shifts observed in NHCACB and NHCA experiments

			i		i-1
Residue	¹⁵N (ppm)	NH (ppm)	C^α (ppm)	C^β (ppm)	C^α (ppm)	C^β (ppm)
99G	118.3	7.96	44.76	NA	61.1	37.8 (98I)^a
110G	127.3	8.21	45.17	NA	61.5	37.6 (109Y)^a
202G	128.3	8.35	45.13	NA	53.3	38.7
201 N	116.4	8.38	53.3	38.5	44.9	29.4
200R	126.8	8.38	44.9	29.7	ND	ND
208G	126.8	8.44	44.9	NA	58.5	64
207S	119.2	8.25	58.5	63.8	61.4	69.3
206T	124.9	8.06	61.35	69.2	52.5	18.5
205A	112.6	8.45	52.4	18.5	56.1	29.6 (204E)^a
217T	120.4	8.26	61.57	69.1	56.04	29.6
216E	114.9	8.4	56.1	29.6	52.85	38.5 (215N)^a
224L	118.4	7.84	61.8	37.5	56.6	29.7
223R	110.6	7.94	56.4	29.8	61.1	37.6
222I	116.4	8.1	61.1	37.7	45	NA (221G)^a
273G	127.5	8.37	45	NA	55.9	32.6 (272M)^a
275R	115.6	8.29	55.97	29.7	52.6	38.2 (274N)^a
282S	121.1	8.18	58.3	63.4	54.2	40.75
281D	118.3	8.105	54	40.7	52.4	18.4
280A	111.1	8.31	52.9	18.65	58.8	63.5
279S	120.7	8.34	58.9	63.4	53	38.4 (278N)^a
309G	126.8	8.38	44.9	NA	56.3	29.7 (308E)^a

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No peaks for its own C^α and C^β resonances were detected.

4. Discussion

Little is known about the three-dimensional structures of transmembrane proteins. Only about 0.5% of the structures in the Protein Data Bank are of transmembrane proteins because of the difficulty of crystallizing membrane proteins [2]. This dearth of structural information signals a need for alternative means for determination of membrane protein structure.

NMR has yet to make a large contribution to the collective knowledge of the complete three-dimensional structures of membrane proteins. Solid state NMR techniques have been used to considerable advantage in the study of membrane proteins [33-40], but no complete membrane protein structures have been reported by solid state NMR except for structures based on one trans-membrane helix. Ordinary solution NMR is handicapped by the lack of solubility in aqueous media of membrane proteins. To circumvent this problem, a large number of studies of membrane protein fragments in both aqueous and organic media have now been reported [41-61]. These studies have provided extensive information on the secondary structure of the fragments, which in turn have been shown to report on the secondary structure of the intact protein. These studies by themselves cannot provide extensive three-dimensional information. However, it has been possible to successfully combine such experimental information of secondary structure with non-NMR experimental distance data to develop moderate resolution structures for entire integral membrane proteins [62].

The greatest success from solution NMR studies so far has been with the bacterial porins, which are formed of β-barrels. Complete three-dimensional structures have now been reported for several porins from high resolution solution NMR data [5,6,63-66]. More recently, backbone assignments of KcsA in SDS micelles [67] and studies on diacylglycerol kinase with three transmembrane segments per monomer have been reported [15].

In this study, we have made some progress in the structure analysis of the SPase I, an integral membrane protein with two transmembrane segments, using solution NMR techniques. The NMR data indicate that it is feasible to obtain structural information on large membrane proteins. The acquired NMR spectra of the intact SPase I show moderate resolution under conditions in which SPase I activity is maintained. Remarkably, considering the size of the SPase I-detergent micelle complex, about 75% of SPase I residues gave rise to resonances in the 2D¹H-¹⁵N HSQC spectrum. Moreover, some of these resonances were assigned, using a combination of HNCACB, HNCA, and HN(CO) experiments of uniformly²H,¹³C,¹⁵N-labeled SPase I (Table 1). This suggests that in addition to the TROSY experiment, which enhances the quality of the observed NMR spectra, application of a more sensitive CBCA(CO)NH experiment, might lead to better resolution of the NMR data and therefore increase the number of assigned residues.

All of the assigned segments of the uniformly²H,¹³C,¹⁵N-labeled SPase I were located in the catalytic extramembraneous domain of the protein. The count of observable Gly residues was consistent with the visualization of only the extramembraneous domain of the protein. Therefore it appears that the NMR spectrum arises almost entirely from the C-terminal extramembraneous domain of the SPase I, with little or no contribution from the transmembrane domain of this protein. There are two possible explanations for this observation. First, the presence of the²H isotope affects the NMR experiments because the peptide backbone of the transmembrane segments (inside the detergent micelle) does not back exchange for¹H due to the stability of the transmembrane α-helices, while the more exposed backbone of the β-sheet in the extramembraneous domain does. Under the simplifying assumption that the amide protons not observed are actually deuterons that have not efficiently exchanged with protons, the NMR data indicate that an upper limit of about 25% of the amides has not exchanged. This number is close to the estimate of about 30% of the protein's residues in the transmembrane domain. This suggestion is consistent with the slow¹H-²D exchange expected from the stable helices of a transmembrane domain in a hydrophobic environment (interior of a detergent micelle). Second, the transmembrane helices within the detergent micelle are conformationally rigid and only experience the correlation time of the rotational diffusion of the detergent micelle, while the extramembraneous domain may experience some independent motion, leading to some additional averaging of dipolar interactions in the latter case. Some improvement might then be expected at higher temperature, which can lead to shorter rotational correlation times of the ensemble. However we found that the protein is degraded substantially at higher temperatures; therefore the temperature was maintained at 22 °C during the time of acquisition. Increasing field strength should increase the resolution and produce better data. However we compared the data from a 900 MHz spectrometer, which is the highest field strength available, with the data from a 600 MHz spectrometer and cold probe, both with TROSY, and observed little improvement in resolution for this protein at the higher field strength. The TROSY effect is modest even at high field and the MAS experiments did not yield significant improvement in linewidths. These observations suggest that in this particular membrane protein, contrary to expectations, internal motions in the extramembraneous domain may play a significant role in determining linewidth and resolution.

NMR chemical shifts have a strong correlation with the secondary structure of a protein [68,69]. The method presented by Wang et al. provides a fast and reliable way to identify secondary structure based on the NMR chemical shifts [70]. They introduced a new approach, called Probability Based Secondary Structure Identification, to identify protein secondary structure from NMR chemical-shift data. To predict secondary structure of identified SPase I segments, C^α was compared to the chemical shifts categorized according to secondary structure type as calculated by Wang et al. We found that most chemical shifts observed for intact SPase I correspond to β-strand or random coil in agreement with the known crystal structure of the SPase I catalytic domain. This analysis is consistent with the SPase I in its native configuration, consistent with the activity data.

Further work on the whole structure of SPase I is in progress. Since detergents other than the DPC used in this study have been shown to provide a better NMR spectrum [71],we are currently examining the activity and stability of SPase I in diverse detergent micelles. In addition, it would be useful to obtain NMR data from SPase I in complex with its inhibitor. The high rate of enzyme autolysis at elevated temperature might be reduced with the presence of the inhibitor, acting as the competitor. Binding of the inhibitor might also stabilize the enzyme, and ensure that the enzyme is in native conformation; consequently NMR experiments might be acquired at higher temperature resulting in better resolved spectra.

Acknowledgements

We thank Klaas Hallenga (National Magnetic Resonance Facility at Madison, University of Wisconsin—Madison) for collection of NMR data on the 900 MHz spectrometer, Gregory Choi (Structural Biology, University of Connecticut) the for help in the collection of NMR data on the 600 MHz spectrometer and Patricia Wilkinson of Bruker BioSpin Corporation (Billerica, MA) for the HRMAS experiments. This research was supported in part by National Institutes of Health Grant GM37639 (to D.A.K.) and in part by National Institutes of Health Grant GM65250 (to P.L.Y.).

Abbreviations

DPC: dodecylphosphocholine
HRMAS: high resolution magic angle spinning
HSQC: heteronuclear single-quantum correlation
NMR: nuclear magnetic resonance
OG: n-octyl-β-D-glucopyranoside
SPase I: type I signal peptidase

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Solution NMR of signal peptidase, a membrane protein

Monika Musial-Siwek

Debra A Kendall

Philip L Yeagle

Abstract

1. Introduction