Intermolecular DetergentâMembrane Protein NOEs for the

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Intermolecular Detergent – Membrane Protein NOEs for the Characterization of Dynamics of Membrane Protein-Detergent Complexes Cedric Eichmann, Julien Orts, Christos Tzitzilonis, Beat Rolf Vögeli, Sean T. Smrt, Justin L. Lorieau, and Roland Riek J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp509137q • Publication Date (Web): 24 Nov 2014 Downloaded from http://pubs.acs.org on November 28, 2014

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The Journal of Physical Chemistry

Intermolecular Detergent – Membrane Protein NOEs for the Characterization of Dynamics of Membrane Protein-Detergent Complexes Cédric Eichmann1*, Julien Orts1*, Christos Tzitzilonis1,2*, Beat Vögeli1, Sean Smrt3, Justin Lorieau3,@, and Roland Riek1,2,@ 1

Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, ETH-

Hönggerberg, CH-8093 Zürich, Switzerland 2

Structural Biology Laboratory, The Salk Institute, La Jolla, California 92037, USA

3

Department of Chemistry, University of Illinois at Chicago, 845 West Taylor St, Room

4500, Chicago, IL, USA 60607 *These authors contributed equally to this work. @

Correspondence should be addressed to R. R. [email protected], +41

44 632 61 39 (tel), +41 44 632 10 21 (fax) and J. L. [email protected], +1 312 355 0488 (tel)

1

ACS Paragon Plus Environment


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Abstract The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the 3D structure and dynamics of membrane proteins. In NMRbased structural studies of membrane proteins qualitative analysis of intermolecular NOEs or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between 1H of the detergent and 1HN of 2Hperdeuterated,

15

N-labeled α-helical membrane protein-detergent complexes following

the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the E. coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

Keywords: Membrane protein, NMR, exact NOEs, eNOE, detergent, dynamics

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Introduction The 3D structure determination of membrane proteins by solution state NMR is usually based on reconstitution of membrane proteins into detergent micelles (1-23), or bicelles (24-27), and into nanodiscs (28-37). For the structure calculation after sequential assignment of the resonances by mostly triple resonance experiments (38-45), the collection of structural restraints include usually paramagnetic relaxation enhancement (18,46), residual dipolar couplings (RDC) (47,48), secondary chemical shifts and protein 1

H-1H NOEs (Nuclear Overhauser Enhancement) (49).

However, structure determination of helical membrane proteins by NMR is still very demanding typically due to the large molecular weight of the detergent-protein complex, the small chemical shift dispersion of the resonances as well as the low number of long range NOEs (in addition to sample preparation problems and sample stability) (50). The latter two bottlenecks are usually attributed to either the large amount of helices present or the small number of aromatic side chains in the core of the protein structure, but also to (functionally relevant) slow conformational exchange dynamics of the protein and a not well-folded tertiary structure of the protein due to not optimal membrane mimicry of the detergent. In this context it is interesting to note that several recent structural studies on helical membrane proteins, including for example the 23-residue full-length fusion domain of the influenza hemagglutinin fusion protein and the hepatitis C virus P7 viroporin (4,7,11,16,17,19,22,51), indicated that the spread of the chemical shift dispersion correlates somewhat with the number of long range NOEs. Along with this correlation it was the notion of the authors that in protein-detergent systems with a small chemical shift dispersion (5,52), the omnipresent intermolecular detergent-protein NOEs were of similar intensity within a transmembrane (TM) helix irrespective of the location of 3



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the amide (5,12,51,53,54), while a more distinct pattern was observed in so called well behaving systems (such as HAfp23). In general, intermolecular NOEs between protein and detergent/lipid resonances are used on a qualitative basis to identify residues, which are involved in the transmembrane segment of the protein (5,18,52,55-57). Here, we investigated the aforementioned observation in detail by applying the recently introduced concept of exact NOEs (eNOEs) (58-60), which enables the accurate determination of the average distance between two protons, to the detergent-protein NOEs of interest both theoretically and experimentally. The following analysis indicates that detergentprotein NOEs are a qualitative probe to measure dynamics of membrane proteindetergent complexes.

Theoretical and Experimental Methods NOE theory Up to date the full relaxation matrix approach is the most accurate method to describe the magnetization transfer between nuclear spins. It can take into account spin diffusion between multiple spins, chemical exchange between multiple states and degenerate spins rigorously within the semi-classical theory frame. The relaxation matrix representing a system composed of protein and detergent under chemical exchange has the following form:

(1)

4


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Where

bound free , and Rderergent are sub-block matrices representing one type of Rprot , Rdetergent

molecule and each diagonal element of these sub-blocks corresponds to a spin of the molecule. The kinetic matrix, K , corresponds to the chemical exchange of the detergent molecules between the bound and the free state. Every block matrix is composed of diagonal elements, the auto-relaxation rates ρ, and the off-diagonal elements, which are the cross-relaxation rates σ, classically derived for a rigid molecule as following:

(2) where γ is the proton gyromagnetic ratio, ω is the spectral frequency of the nuclei, µ0 is the permeability in vacuum, and ħ denotes Planck’s constant. rij is the internuclear distance in a hypothetically rigid structure and τc is the rotational correlation time of the molecule assumed to be rigid. Solving the Solomon equation (61) we can now derive the NOE intensity between any spin of the system: (3) This expression is difficult to be used in a practical manner. Hence, as we shall see for the purpose of this study, crude approximations are made and their validity is discussed. This includes that the transfer of magnetization between the protein and the detergent

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protons is described with a two spin approximation following the formalism by Vögeli et al. (58) for the eNOE, while spin diffusion, detergent chemical exchange, and degenerated detergent spins can be inferred with this assumption following an adapted formalism by Orts et al. (62), which includes in the established full relaxation matrix approach (63-65) chemical exchange (66-68). First, we recall the theory for the extraction of (intermolecular) effective distances from NOE cross-relaxation rates between detergent and protein protons without chemical exchange. The cross-relaxation rate between a detergent 1H and a protein 1H (such as the 1HN used in the following) can be derived from the cross peak and diagonal peak intensities from a series of multi-dimensional NOESY experiments following the two spin approximation (61,66,69,70). This approach is possible if potential spin diffusion is limited by the selection of short mixing times and protein perdeuteration (see also below). The solution of the two spin Solomon equation is then given by

(4)

∆S z ( t ) σ IS  e − λ t − e− λ t  =−  ∆I z (0) (λ + − λ − ) −

(5)

+

with

λ± =

(ρ I + ρ S ) 2

2

 ρ − ρS  2 ±  I  + σ IS 2  

(6)

of the dipolar coupled 1/2 spins I and S. I denotes the 1H of the detergent and S is the protein 1H, respectively. ρX is the auto-relaxation rate of spin X and σIS the crossrelaxation rate between spins I and S. The simplest and most common way to extract distances from the measured crossm relaxation rate is to introduce an effective cross-relaxation rate σ eff under the

6


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assumption of a rigid molecule. Local rotational motions are absorbed into an order parameter S2, (7)

.

Note that this holds true for motions faster than the overall tumbling of the proteindetergent complex as well as for cases where J(2ω) cannot be neglected. Introduction of chemical exchange into this formalism in a simple form requires the assumption that the exchange rate is very fast, typically by at least two orders of magnitudes faster when compared to the relaxation rate. Under this condition, it has been previously shown that the observed transferred NOE cross-relaxation rate can be accurately modeled as a population weighted average of the cross-relaxation rates for the bound and free form (71,72). In the case of the protein-detergent NOE, the crossrelaxation in the free state is zero. Thus, the effective cross-relaxation rate is simply scaled by the population of the protein that is bound to lipid or detergent molecules, pb: (8) In other words an exchange rate of 1 kHz or 1 MHz will not make a difference in a typical NOE build-up curve because the chemical exchange rate is now in the so-called fast regime (Suppl. Figure S1). Subsequently, as shown in the Supplementary Material not only the chemical exchange rate influences the bound population of the detergent but also the kinetic model used to fit the data (Suppl. Figures S2, S3). In our study, this more complex analysis was not required because a large excess of detergent molecules was used to drive the bound population to 1.0 (Suppl. Figure S3). This conclusion is furthermore supported for HAfp23 by the high hydrogen exchange protection factors, which indicate a tight binding of HAfp23 to the detergent micelle (51).

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An additional complexity appears in the case of cross-relaxation rates between detergent molecules and the protein. Several detergent molecules are close in space to the amide

1

H spins of interest and every detergent molecule has a number of

degenerate protons with identical 1H chemical shifts, including in particular most of the methylene resonances. Hence, a large number of 1H spins from the detergent may take part in the measured cross peak build-up, thereby influencing the initial magnetization, ∆IZ(0), as well as the effective internuclear distance, reff, defined as follows:

(9) in which N is the sum over the detergent 1H spins with degenerate chemical shifts. Finally, the following effective NOE cross-relaxation rate is obtained:

(10) Simple model for the evaluation of detergent-protein NOE cross-relaxation rates In order to model with a highly simplified approach a NOE cross peak build-up between a degenerate detergent methylene 1H with a non-degenerate protein 1HN amide proton in an otherwise deuterated detergent-embedded model α-helix, the following assumptions were made based on geometrical considerations: (i) A poly-Leu α-helix was selected to keep the system as simple as possible. The same qualitative results are obtained for a poly-Ala α-helix (Suppl. Figure S4). (ii) Detergent methylene protons are mass centered at a distance of 7.4 Å from the center of the α-helix. (iii) The detergent methylene protons are evenly distributed at a distance of 2.5 Å between each other reflecting both the intramolecular distance between methylene protons within a detergent chain (which is 2.2 Å), as well as the average distance between two detergent 8


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molecules. The location of these detergent protons in Figure 1A are highlighted in two dimensions. (iv) Only the first layer of methylene protons around the α-helix is taken into

( )6

account for the magnetization transfer because of the short-distance 1 reffXY

relationship of the NOE. (v) It is assumed that the local intermolecular order parameter S2 between all detergent protons and the amide proton of interest are identical. This assumption is based on the notion that the detergent molecules behave dynamically all the same in a detergent-protein complex excluding thereby detergent molecules that bind tightly to the protein in a lipid-binding site, for example. (vi) 1HN positions are located at ideal positions within the α-helix as highlighted in Figure 1A with the α-helical wheel-type character. Following

these

assumptions,

the

detergent-protein

NOE

pattern

within

a

transmembrane helix in a multi-span membrane protein was calculated for various extents of detergent accessibilities from 180°, indicating that the helix of interest is packed to other transmembrane helices, up to 360° for an α-helix surrounded completely with detergent (Figure 1B - E). The same calculations were made for a polyAla α-helix having the detergent molecules mass centered at a distance of 5.4 Å from the center of the α-helix because of the shorter side chains present (Suppl. Figure S4).

Molecular dynamics simulations and numerical calculation of lipid-protein NOEs In order to get a more realistic distribution of detergent/lipid protons surrounding a transmembrane helix than described by the simplified model above, very short molecular dynamics (MD) simulations were performed for a single poly-Leu α-helix to penta polyLeu α-helices embedded in a model lipid bilayer. These simulations have only been

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done for the calculation of detergent-protein NOEs without an attempt to simulate the dynamics of such a model system. The detergent-protein NOEs were calculated from these models including the possible effects of spin diffusion, exchange, and dynamics. Model buildings: The atom coordinate files (pdb format) of poly-Leu α-helices, 30 amino acid residues in length each, used in the models were created with Chimera (73) and manually adjusted to form a dimer, tetramer, and pentamer mimicking thereby a two, four, and five transmembrane helical packed membrane protein, respectively. The 3D structure files (psf) were created with VMD (74). The membrane bilayer is made of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. The various structures were embedded in the membrane manually and solvated with VMD. Clashing molecules (i.e. water with lipid molecules) were removed with VMD. The final volume of the system was a cuboid of ~753 Å3. Equilibration of the systems with molecular dynamics: The lipid tails were melted with constant pressure of 1 bar at a temperature of 300 K. 250,000 steps were performed during the simulation with 2 fs/step, using the CHARMM force field (75-79) keeping the protein, the water molecules, and the polar moieties of the lipids fixed. Periodic boundary conditions and a particle mesh Ewald grid for full electrostatic evaluation were employed. Subsequently, the equilibration period was further extended by 250,000 steps with the protein constrained to its initial position using a harmonic potential with a force constant of 1 kcal.mol-1 Å-2 while the lipid and the water molecules were remained free. Finally, an apparent equilibration with the protein released from the constraints was performed for 250,000 steps for the poly-Leu α-helices. All simulations were run at constant temperature and constant pressure with the Nosé-Hoover Langevin piston (80,81) and the Langevin dynamic (81). Water molecules were kept outside the 10


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membrane during equilibration. The snapshots of the equilibrated system MD trajectory were used for the analysis. NOE calculations: NOEs between amide protons of the protein and methylene protons of the lipids were calculated using the full relaxation matrix approach (62-68). The perdeuterated protein is protonated only on the exchangeable protons including the backbone 1HN protons. Every proton participating in the NOE directly or indirectly via spin diffusion was considered as one element in the matrix and no pseudo atoms were used. The individual contribution to the NOE from each of the degenerate methylene protons was thus added together. Furthermore, it was assumed in this study that the exchange rate of the detergent molecules is fast compared to the relaxation rates (71,72) (i.e. ‘fast regime’) and the kinetics are similar for all detergent molecules contacting the protein (i.e. ‘homogenous kinetic’). In addition, an isotropic rigid body spectral density function was used to calculate the auto-relaxation and cross-relaxation rates. More details are given in the Supplementary Material. The rotational correlation tumbling time of the complexes was set to 17.4 ns for the model systems composed of poly-Leu α-helices. The selected magnetic field was 700 MHz 1H frequency. The NOEs were calculated for mixing times of 10, 60, 120, 240, and 300 ms. The initial magnetization was set to 1.

Expression and purification of the influenza HAfp23 peptide The 23-residue full-length 2H,

13

C,

15

N-labeled influenza HAfp23 peptide was prepared

as previously described by Lorieau et al. (51). 2H, 13C, 15N-minimal M9 medium (83), 1 L, was inoculated with a starter culture grown from the E. coli strain BL21(DE3) with the pET-11a vector including GB1-HAfp23, and incubated at 37 °C while shaking at 250 rpm. 11



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The cell culture was induced with 2 mM IPTG (Invitrogen) at a cell density of OD600 = 0.5. After 4 hours of expression, the cells were pelleted by centrifugation at 8,000 g for 1 hour. The pellet was resuspended in 25 mM Tris-HCl supplemented with 8 M urea at pH 7.4, sonicated and purified with a HisTrap column (GE Healthcare) followed by elution with 220 mM Imidazole. The sample was dialyzed against 25 mM Tris-HCl pH 7.4 and further purified by size exclusion chromatography before cleavage with 7 µg of FactorXa per 1 mg substrate (Haematologic Technologies). The final peptide product was purified by reverse phase chromatography (GE Healthcare) and lyophilized. The NMR sample was prepared by resolubilization to a final concentration of 0.9 mM into 25 mM 2

H-Tris-HCl pH 7.4 buffer containing 135 mM protonated dodecylphosphocholine (1H-

DPC, Sigma) and 6.25 % D20. Protein and lipid concentrations were quantitated from peak integrals in a 1H 1D NMR experiment.

Expression and purification of the E. coli inner membrane protein YidH The pET3a-LIC based plasmid containing the full-length YidH (SwissProt ID P0ADM0) gene was provided by Robert Stroud’s lab at UCSF (84). YidH was expressed in the E. coli strain BL21(DE3) pLysS Star (Invitrogen, Carlsbad, USA). 2H,

13

C,

15

N-labeled YidH

was produced on standard M9 minimal medium (83) based on 2H water (Isotec) and supplemented with 1 g/L of

15

NH4Cl (Isotec) and 2 g/L

13

C Glucose (Isotec). Cells were

grown at 37 °C until reaching an OD600 = 0.8 and transferred to 18 °C for 45 min. YidH was expressed by induction with 0.5 mM IPTG (Invitrogen) at 18 °C for 12 hours. For a perdeuterated growth all the buffers were made with 2H and deuterated

12

C or

13

C

Glucose (Isotec). This approach results in a nearly perdeuterated sample with exception of the exchangeable backbone amide protons and amine protons in the side chains. 12


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After expression, cells were centrifuged at 5,000 g for 10 minutes and resuspended in the lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM DTT, 0.5 mg/ml lysozyme (Sigma), protease inhibitor cocktail tablets (Roche)) and incubated at 4 °C with gentle stirring for 30 minutes. Cells were lysed by two Microfluidizer (Microfluidics) cycles at 80,000 psi. The lysed cells were centrifuged at 8,000 g for 15 minutes. The supernatant was collected and centrifuged at 100,000 g for 2 hours. The pelleted membrane fraction was resuspended in extraction buffer, 50 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM Imidazole, supplemented with 20 mM

1

H-DPC, 5 mM TCEP (Tris(2-carboxyethyl)

phosphine), 10 % v/v Glycerol and protease inhibitor cocktail tablets (Roche). YidH was extracted over night by gentle stirring at 4 °C. The solubilized membrane protein fraction was separated from large aggregates by centrifugation at 100,000 g for 45 minutes. The supernatant was loaded on a 5 ml Nickel Sepharose 6 Fast Flow resin (GE Healthcare), pre-equilibrated with 50 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM Imidazole, 3 mM 1HDPC and 1 mM TCEP. The resin was washed with the latter buffer, followed by elution of YidH with 50 mM Tris-HCl pH 8, 300 mM NaCl, 500 mM Imidazole, 3 mM 1H-DPC and 5 mM TCEP. Fractions containing the protein were pooled together and the buffer was exchanged using a PD10 desalting column (GE Healthcare) to 20 mM Bis-Tris-HCl pH 7, 3 mM 1H-DPC and 5 mM TCEP. For the sample preparations of YidH with DHPC7 (1,2-diheptanoyl-sn-glycerol-3-phosphocholine) and LMPG (1-myristoyl-2-hydroxy-snglycero-3-[phospho-rac-(1-glycerol)]) micelles, the extraction buffer contained 15 mM DHPC-7/1 mM LMPG, washing buffer 3 mM DHPC-7/1 mM LMPG, and the elution and desalting buffer 6 mM DHPC-7/1 mM LMPG. For NMR measurements YidH was concentrated not more than 10 times (85) using a 50 kDa (for the DPC sample) and 30 kDa (for the DHPC-7/LMPG sample) molecular weight cut-off Centricon (Amicon) 13



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concentrator to a final sample concentration of 0.3 - 0.5 mM in the aforementioned buffer conditions with either ca. 30 mM 1H-DPC or 60 mM DHPC-7/10 mM LMPG. 3 % of D2O was added to the final sample for the NMR measurements.

NMR spectroscopy and analysis NMR experiments were performed on a Bruker 700 MHz spectrometer equipped with a triple resonance cryoprobe at a temperature of 305 K for HAfp23 and 314 K for YidH, respectively. All spectra were processed with the program PROSA (86) and analyzed with the program XEASY (87). Peak intensities were determined by taking the maximal peak height rather than integrating volumes following the procedure described by Vögeli et al. (2009) (58). For HAfp23 at pH 7.4, 3D

15

N-resolved [1H,1H]-NOESY experiments were acquired with

NOE mixing times τm = 52, 77, 102, 152, and 202 ms for the determination of the NOE build-up rates. DPC-protein 1HN NOE cross peak intensities from a single mixing time τm = 100 ms have also been measured for HAfp23 at pH 4 (and published in ref. 51) at which HAfp23 starts to interchange rapidly between the major tight and a minor open structure populated to about 20 - 28 % (82). Furthermore, DPC-protein 1HN NOE cross peak intensities from a single mixing time τm = 100 ms have been measured for HAfp20, which is three residues shorter at the C-terminus than wild-type and shows an open structure with a population of about 90 % (88). The sequential assignments of YidH in both detergents used were obtained by 3D TROSY-HNCA (89), 3D TROSY-HNCACB (90), 3D TROSY-HNCOCA (90), and 3D 15Nresolved [1H,1H]-NOESY experiments (91). The 3D

15

N-resolved [1H,1H]-NOESY

experiments were also used to measure NOE build-up rates. For YidH, experiments 14


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were acquired with NOE mixing times τm = 32, 47, 62 ms for both detergents and in the case of the DHPC-7/LMPG mixture the additional mixing time 152 ms was measured. Following the studies by Vögeli et al. (2010) (59) the NOESY element was placed before the HMQC element in the 3D

15

N-resolved [1H,1H]-NOESY experiment in order to

suppress the strong residual water and detergent signals. The corresponding intensity of the detected magnetization transfer from N degenerate detergent protons 1HD,i to 1

HP of the protein originating from the initial detergent magnetization with intensity

can be expressed as follows,

(11) where

accounts for the part of the detergent magnetization that has recovered

during the interscan delay. element and

denotes the loss of magnetization during the HMQC

is described by equation 4. Considering that the detergent protons

relax all similarly and are all degenerate (i.e. the methylene protons), an effective NOE transfer is defined as follows

(12) yielding a simplified equation 11: (13) where

accounts for the part of the detergent magnetization that has recovered

during the interscan delay. This value was experimentally determined for a relaxation delay of 1.0 s used in the NOESY experiments by a series of 1D 1H NMR experiments with variable interscan delays. With the same experiments

15


was determined.


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denotes the loss of magnetization during the HMQC element, and

Page 16 of 50

is described

by equation 12 using the effective distance defined previously. Note, if the peak intensity is determined by the maximum height rather than volume this procedure may translate into an additional correction term that is however absorbed by

. The term of

can be extracted if also the diagonal peak intensity at τm = 0 is either

interest

measured or back predicted: (14) with and

(15) Note, with this approach the interscan delay must be chosen such that

is

approximately 1 or should be experimentally determined. An exponential fit of the diagonal further allows also the determination of ρP, which together with the determined

ρD from 1D experiments enables the determination of an effective cross-relaxation rate eff from the cross peak build-ups straight forwardly using a one parameter fit following σ DP

equation 5. T1 and T2 relaxation times of the backbone

15

N nuclear spins of the YidH-detergent

complex were measured using standard pulse sequences (92). The global correlation time τc was calculated from the ratio T2/T1 (93) using the program Modelfree4 (94,95) under the assumption of an isotropic overall tumbling. The correlation time was crosschecked with the NOE build-up rates of 1HN-1HN NOEs within an α-helix of YidH following Vögeli et al. (2010) (59). In the case of HAfp23 the rotational correlation time

16


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has been published elsewhere (51,82). Furthermore, 13C natural abundance T1 = 2.02 s and T2 = 0.24 s measurements for DPC in complex with HAfp23 were obtained to estimate the local order parameters of the detergent. As discussed in the main text, there is under certain circumstances also the possibility to extract the helical-wheel pattern of the detergent-protein NOEs from a single 3D

15

N-

resolved [1H,1H]-NOESY spectrum. The extracted NOE cross peaks must however be normalized to the diagonal cross peak. For very long mixing times however, it is suggested to normalize the detergent-protein NOE by the sum of the intensities/volumes of all cross peaks and the diagonal peak for a given 15N-1H-moiety. Saturation transfer 2D [15N,1H]-TROSY experiments saturating during the interscan delay either the methylene (at 1.25 ppm for HAfp23 and YidH in complex with the detergent DPC and the DHPC-7/LMPG detergent mixture) or the water resonance (4.68 ppm for HAfp23 and 4.59 ppm for YidH) (Suppl. Figure S8) have been carried out as described by Chill et al. (2006) (96).

17



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Results and Discussion Modeling of detergent-protein NOEs In order to get insights into an expected pattern of detergent-protein NOEs along a transmembrane α-helix of a membrane protein-detergent/lipid complex, we modeled the corresponding intermolecular cross-relaxation rates between degenerate detergent protons to non-degenerate 1HN resonances. For simplicity we assumed a perdeuterated 18-residue long poly-Leu α-helix embedded at various degrees of detergent micelles following several assumptions mentioned in the theory part. As highlighted in Figure 1A, a set of detergent 1H labeled D1 – D17 surrounds the α-helix at a distance of 7.4 Å from the center of the helix. The 1HN protons labeled 1 - 18 are located on the α-helix-typical wheel at a distance of 1.6 Å from the center. If the poly-Leu α-helix is embedded completely into the detergent, the intermolecular NOE cross-relaxation rates and concomitantly the normalized NOE cross peak intensities (i.e. under the assumption that all 1HN protons of interest have similar T1 and T2 relaxation times) show the same intensity along the amino acid sequence (Figure 1E). In contrast, if the poly-Leu α-helix is partially not interacting with detergent molecules, a helical-wheel alteration of NOE cross-relaxation rates starts to emerge (Figure 1C,D). If the helix is only half surrounded (i.e. 180° or 50 %, respectively) by detergent molecules (i.e. D1 – D9 in Figure 1A), a strong helical-wheel type alteration of NOE cross-relaxation rates along the amino acid sequence is calculated (Figure 1B). This finding is expected since 1HN numbered 1, 8, and 12 are much closer to the detergent 1H localized at position D1 – D9 than 1HN 3, 10, and 17, respectively. The helical-wheel NOE pattern is highly pronounced because of the inverse sixth power proportionality between the relevant distances and the NOE (equation 2). Furthermore, the qualitative nature of this alteration does not change by 18


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other distances between the

1

HN amide protons and the detergent protons as

exemplified by a poly-Ala α-helix (Suppl. Figure S4). Please note that the presented model calculations are only able to give a qualitative picture of the presence of the helical-wheel character of the detergent-protein NOEs discussed and not a quantitative one because of the lack of internal motion (i.e. S2 in equation 10), the lack of taking into account the bound population (i.e. pb in equation 10), the simplifcation to a poly-Leu αhelix, and the use of a simplified detergent/lipid along with a somewhat inaccurate description of the 1H spin density of the detergent/lipid molecules. Figure 1

Figure 1: A simple poly-Leu α-helix model surrounded only partially by detergent/lipid molecules indicates the presence of a helical-wheel type pattern of detergent/lipid protein NOEs along a transmembrane αhelix. Simulated detergent-protein NOE crossrelaxation rates (y-axis) between the amide moieties of a poly-Leu α-helix (x-axis) embedded to varying degrees in a simplified detergent micelle/membrane. (A) The simulation is based on the geometrical considerations given in the cartoon. The amide moieties labeled with numbers 1 - 18 are located on the helical wheel with a radius of 1.6 Å. The detergent protons labeled with D1 - D17 are located on the cylinder with radius 7.4 Å dictated by the size of the poly-Leu α-helix and are 2.5 Å apart from each other. Indicated by dashed lines are the extents of detergent surrounding the α-helix for the calculations in B - E. (B E) Following the cartoon in (A), relative NOE crossrelaxation rates were calculated for a 50 % (B), 71 % (C), 82 % (D), and 100 % (E) detergent embedded poly-Leu α-helix in the absence of spin diffusion. The rates are relative because the simulation lacks knowledge about the dynamics of the detergent, the population of detergent on the helix, and the exact position of the detergent molecules relative to the protein. In the linear regime (i.e. in the absence of spin diffusion), the presented graphs also recapitulate the relative changes of the normalized detergentprotein NOE intensities in the NOESY experiment along the helix. 19



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To strengthen the findings on the helical-wheel pattern of the NOEs from the simplified model described above, short MD simulations on poly-Leu α-helices embedded in a model lipid bilayer were calculated (note, a lipid bilayer of POPC was used, since a lipid patch and a force field were available). This approach has been taken in particular to describe the 1H spin density of the lipids more accurately than with the simple model introduced above (note: it is not the attempt to sample the dynamics of the membrane protein complex). As shown in Figure 2, calculations were performed on a single polyLeu α-helix embedded entirely in the model membrane, two interacting poly-Leu αhelices representing a two transmembrane spanning membrane protein, as well as four and five interacting transmembrane poly-Leu α-helices representing more complex 3D structures of membrane proteins. For all simulations intermolecular eNOEs between 1HN of the blue helix with methylene lipid protons were calculated by a software following the same formalism as implemented in eNORA (62), which is based on a full relaxation matrix approach that takes into account spin diffusion as well as chemical exchange. In order to enhance the sampling of the NOE cross-relaxation rates and normalized cross peak intensities, 50 snapshots of the MD trajectory were averaged arithmetically. However, an explicit treatment of (fast) dynamics was not done. Figure 2A shows the calculated NOE pattern along the poly-Leu α-helix embedded in the model lipid bilayer. The helical termini show smaller NOE cross-relaxation rates than the central segment attributed to the location at the water-bilayer interface. Furthermore, the distribution of the NOE cross-relaxation rates is less homogeneous than expected, which is attributed to an insufficient ensemble sampling. Nonetheless, the central segment shows all similar NOEs. In contrast, a helical-wheel pattern of NOE cross-relaxation rates can be 20


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observed qualitatively if the blue α-helix is interacting with a second helix (Figure 2B). This effect gets much more pronounced if the blue helix is embedded in a more complex 3D structure composed of four or five packed transmembrane α-helices as exemplified in Figures 2C and 2D reflecting nicely the results from the simple model system introduced above (Figure 1). While the helical wheel pattern can be recognized easily by eye (Figures 1 and 2) one may ask for the presence of the pattern from a quantitative perspective. It is suggested that the detergent-protein NOE pattern can be evaluated by inspection of the maximal difference of the detergent-protein NOEs for amino acid residues, which are at least three and at most four residues apart from each other in the protein sequence. If this difference is larger than the standard deviation of all detergent-protein NOEs measured within a helix and larger than the signal to noise of the measurement a helical-wheel NOE pattern can be assumed to be present. In summary, based on theoretical considerations the detergent-protein NOE pattern along the amino acid sequence of a helical membrane protein is of helical wheel-type reflecting thereby the detergent/lipid accessibility of a transmembrane or a membraneattached helix in detail.

21



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Figure 2

Figure 2: Lipid-protein NOE cross-relaxation rates extracted from simulations of αhelical poly-Leu models embedded into a lipid bilayer and surrounded by 1 - 4 α-helices indicate the presence of a helical-wheel pattern of lipid/detergent protein NOEs along a membrane α-helix. Simulated lipid-protein NOE cross-relaxation rates (y-axis) between amide moieties (x-axis) of a single poly-Leu α-helix (x-axis), color coded in blue in the cartoon, embedded in a simplified lipid bilayer system and surrounded by (A) none, (B) one, (C) three, or (D) four other poly-Leu α-helices as indicated (see main text for more details). The rotational correlation time of the molecule was assumed to be 17.4 ns. In the linear regime, the NOE cross-relaxation rates are proportional to the corresponding normalized NOE cross peak intensities (see Figure 5). 22


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Detergent-protein NOE cross-relaxation rates of the complete influenza hemagglutinin fusion domain HAfp23 The recent structural studies by Bax and coworkers (51) on the perdeuterated membrane-attached 23-residue peptide influenza hemagglutinin fusion domain (HAfp23) bound to protonated DPC detergent micelles at pH 7.4 revealed a remarkably tight helical hairpin structure with the N-terminal α-helix (residues 1-12) packed against the second

α-helix

(residues

14-23)

(Figure

6A,

inset).

Furthermore,

qualitative

interpretation of detergent – protein 1HN NOE data (ref. 51, Figure 6A) and saturation transfer experiments (Suppl. Figure S8A) indicate that HAfp23 is located at the waterlipid interface, with its hydrophobic surface facing the lipid environment and the Gly-rich site of the helix-helix interface exposed to the solvent (51). At pH 4 HAfp23 starts to interchange rapidly between the major tight and a minor open structure populated to about 20 - 28 % (82). The open structure of the variant HAfp20, which is three residues shorter at the C-terminus than the wild-type, is populated to about 90 % (Figure 6C inset) (82). Because of the available 3D structure(s) and the proposed large conformational changes under certain conditions, this system was investigated here in respect to a quantitative analysis of the DPC – protein NOE cross-relaxation rates. DPCprotein NOE build-ups were measured successfully for HAfp23 in complex with 1H-DPC at pH 7.4 (Figure 3A-D) yielding the expected helical-wheel character of DPC-protein NOE cross-relaxation rates (between residues 4-11 and 14-22) as evident from an inspection of Figure 3E.

23



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Page 24 of 50

Figure 3

eff Figure 3: Effective cross relaxation rates σ DP of HAfp23 at pH 7.4 between the methylene protons of the DPC micelle and the backbone 1HN. (A – D) Representative NOE build-ups versus mixing time τm and their fits are shown. (E) Effective crosseff relaxation rates σ DP between the -CH2-groups of DPC and the backbone 1HN of HAfp23 are displayed as an amino acid residue bar plot. The α-helices determined by NMR (51) are depicted above the graph by open rectangles.

24


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Detergent-protein NOE cross-relaxation rates of the membrane protein detergent complex YidH in DPC micelles To expand the finding of the helical-wheel type detergent-protein NOE pattern on HAfp23 (see above) the method was applied also to the membrane protein YidH. YidH is a small E. coli inner membrane protein with 115 amino acid residues and has been predicted by the membrane protein topology prediction method TMHMM (97) to have three transmembrane (TM) helices. When extracted from the membrane and reconstituted into 1H-DPC detergent micelles it is a dimer (C. E., C. T., R. R. not published), and based on helical NOEs and secondary chemical shifts YidH is composed of three TM helices comprising residues 21-38 (TM1), 54-66 (TM2), and 94114 (TM3; C. E., C. T., R. R. not published). The rotational correlation time is 17.4 ns as determined from

15

N relaxation measurements (Suppl. Figure S5). The presence of TM

helices is further supported by the presence of detergent-protein NOEs (Figure 4) and saturation transfer experiments from the methylene detergent protons (at 1.25 ppm ω1(1H)) to the protein (Suppl. Figure S8B). The detailed eNOE analysis from NOE buildup curves (Suppl. Figure S6) of the methylene detergent 1H – 1HN amide NOEs of 2Hperdeuterated, 15N-labeled YidH along the amino acid sequence is given both as a linear as well as a helical wheel-type representation in Figure 4. While the NOE crossrelaxation rate pattern within TM1 is rather uniform, there is a typical helical-wheel pattern along TM2 with small NOE cross-relaxation rates for residues Phe62 and Ser63 on TM2. While the helical-wheel pattern is also not obvious in TM3, there might be a short wheel pattern between residues 103 – 110, and there are three residues with small or zero detergent-protein NOE cross-relaxation rates (i.e. Ile99, Val105, Met108). Following the theoretical findings above, these data indicate that TM1 is surrounded 25



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Page 26 of 50

mostly by detergent molecules, while both TM2 and TM3 are protected from the detergent around residues 62, 63, 99, 105, and 108, respectively, which may indicate the presence of interhelical contacts around the residues mentioned. In line with this finding are preliminary structural studies of the YidH-1H-DPC detergent complex indicating that residues 62 and 63 on TM2 are part of a helix-helix interaction motif within Leu61-Phe62-Ser63-Gly64-Gly65 (C. E., C. T., R. R. not published). In contrast, TM1 is almost entirely surrounded by detergents or/and may rotate extensively around its axis. In summary, based on the detergent-protein NOE cross-relaxation rates, part of the 3D structure of YidH composed of three TM helices appears to be of a highly dynamic nature.

Figure 4

Figure 4: Experimentally derived detergent-protein NOE cross-relaxation rates of YidH in DPC micelles from NOE build-ups indicate the presence of large motion in YidH. eff Effective cross-relaxation rates σ DP between the -CH2-groups of DPC and the backbone 1 N H of YidH are displayed for the first (A), second (B), and third (C) transmembrane αhelix of YidH as amino acid residue bar plots and α-helical wheel representations (G – I). 26


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No assignments were obtained for Met101 and Val102 colored in gray. In the panels (G – I), blue dots indicate an intermolecular NOE exchange cross peak between H2O and 1 N H of YidH. Cross peaks between DPC and 1HN of YidH are shown by purple, red, and green dots for the head methyl, the methylene and the choline methyl protons of DPC, respectively. The absence of intermolecular NOEs is indicated by dots on the inner circles in the corresponding color code. Normalized detergent-protein NOE intensities between the -CH2-groups of DPC and the backbone 1HN of YidH at a mixing time τm = 62 ms for the three transmembrane helices are shown in panels (D – F). Influence of the order parameter S2 on the protein-detergent/lipid NOEs A major source of NOE rate attenuation is the order parameter S2 as depicted in equation 10. The presence of a small order parameter may quantitatively reduce the NOE and NOE cross-relaxation rates proportionally but will not qualitatively change the helical wheel-type pattern. It is expected that the intramolecular order parameter S2 (equation 10) is in the range of 0.1 - 0.2 since intramolecular lipid order parameters in a membrane-like environment have been shown to be in the vicinity of 0.2 (98) and

13

C

natural abundance T1 and T2 measurements for 1H, 13C moieties of DPC in complex with HAfp23 combined with the rotational correlation time of the protein-detergent complex determined from

15

N relaxation measurements indicate that the intramolecular S2 has a

value of ca. 0.17 (data not shown).

A possible simplification: from NOE cross-relaxation rates to normalized NOE cross peak intensities In an attempt to simplify the determination of the detergent-protein NOE cross-relaxation rates by measuring and analyzing several 3D

15

N-resolved [1H,1H]-NOESY spectra, it

has been studied next whether the above helical-wheel type pattern of NOE rates can already be obtained by analyzing detergent-protein cross peaks from a single 3D

15

N-

resolved [1H,1H] NOESY spectrum. Indeed, as exemplified in Figure 6A for HAfp23 27



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Page 28 of 50

normalized detergent-protein NOE intensities measured at a mixing time of 152 ms show a similar pattern as the corresponding NOE cross-relaxation rates (Figure 3E). For YidH an analogous finding is documented. Normalized detergent-protein NOE intensities (Figure 4D - F) measured at a mixing time of 62 ms show a similar pattern as the corresponding NOE cross-relaxation rates (Figure 4A - C). Further support for this simplification is given by the analysis of the MD simulation of the four poly-Leu helical bundle shown in Figure 5. Under the assumption of a rotational correlation time of the molecule of 17.4 ns, the normalized lipid-protein NOE cross peak intensities were calculated along the amino acid sequence for the NOE mixing times 60 - 300 ms. While the helical-wheel pattern gets weaker with longer mixing times attributed to spin diffusion, it is still pronounced up to mixing times of 240 ms (Figure 5C), but obviously shorter mixing times show a more distinct pattern (Figure 5). From this analysis it is predicted that if the product between rotational correlation time and NOE mixing time is not larger than approximately 2 - 3 x 10-9 s2, the analysis of the detergent-protein intensities in a single 3D

15

N-resolved [1H,1H]-NOESY spectrum enables insights into the extent of

detergent exposition of a helix and vice versa, how well the helix is packed in the interior of the protein. These considerations thus indicate that the helical-wheel type pattern of detergent-protein NOE cross-relaxation rates along a transmembrane or membraneattached helix can also be observed using a single data set by listing along the amino acid sequence the intensities of the normalized detergent-protein NOE cross peaks (Figure 5). This reduces the necessary measuring time significantly, simplifies the analysis, and concomitantly can be collected or/and analyzed from almost all membrane protein detergent complexes if available as a deuterated species.

28


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Figure 5

Figure 5: Calculated normalized lipid-1HN protein NOE intensities along the amino acid sequence of the blue poly-Leu α-helix located in a four helical bundle (indicated) embedded in a model lipid bilayer having a rotational correlation time of 17.4 ns. The NOE intensities are given for the mixing times (A) 60 ms, (B) 120 ms, (C) 240 ms, and (D) 300 ms, respectively.

29



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Detergent-protein NOEs of the complete influenza hemagglutinin fusion domain HAfp23 at pH 4 and the variant HAfp20 At pH 4 for which HAfp23 undergoes microsecond exchange dynamics between the tight and a 20 – 28 % populated open structure (82), the helical-wheel pattern of the detergent-protein NOEs is partially smoothened along helix 1 (residues 3-10) when compared to the pH 7.4 data (Figure 6A,B) (82). This smoothing effect could be interpreted as a consequence of the helix-helix opening. To strengthen this hypothesis, normalized DPC-protein NOEs were determined from the variant HAfp20, which has been suggested to be in the open state with a population of about 90 % (88). Indeed, the helical wheel pattern along helix 1 is attenuated significantly (Figure 6C). The remaining pattern is similar to the calculated one for a helix that is approximately 70 – 80 % surrounded by detergent/lipid molecules (Figures 1C,D and 2B) reflecting the membrane-attached nature of the helix.

30


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Figure 6

Figure 6: Normalized NOE intensities between the methylene protons of the DPC micelle and the backbone 1HN of HAfp23 and HAfp20 indicate the presence of the opening of the 3D structure by lowering the pH or by the deletion of C-terminal residues. Experimentally derived normalized DPC - 1HN protein NOE intensities of HAfp23 at pH 7.4 (A), at pH 4 (B), and of HAfp20 at pH 7.4 (C) (51,82,88). Insets of the 3D NMR structures for HAfp23 (A) and the conformational ensemble of the HAfp23-G8A mutant (C) are shown, which analogously to HAfp20 is populated predominantly by the open structures. HAfp23 at pH 4 and HAfp20 at pH 7.4 populate open conformers to 20 28 % and 90 %, respectively, with open structures similar to those shown for HAfp23G8A, though the C-terminal helix of HAfp20 is partially unfolded (51,82). Alongside with the opening of the 3D structure is the partial loss of the helical-wheel pattern of the DPCprotein NOEs.

31



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Detergent-protein NOEs of the membrane protein detergent complex YidH in mixed DHPC-7/LMPG detergent micelles Detergent protein NOEs have also been studied for the membrane protein detergent complex YidH in the DHPC-7/LMPG detergent mixture. The chemical shift analysis of the sequential assignment obtained (albeit less complete than in the presence of 1HDPC) indicates again the presence of three TM helices comprising approximately residues 21-38 (TM1), 54-66 (TM2), and 94-114 (TM3), respectively (C. E., C. T., R. R. not published). The rotational correlation time τc of the protein-detergent complex determined by helical 1HN-1HN eNOEs (59) is 18.3 ns, which is also similar to the YidH – 1

H-DPC detergent complex (τc = 17.4 ns). However, the two systems studied vary

significantly in their detergent-protein NOEs. While in the case of YidH in complex with 1

H-DPC many strong detergent-protein NOE cross peaks are observed in the 3D

15

N-

resolved [1H,1H]-NOESY spectrum, detergent-protein NOE cross peaks are absent in the YidH DHPC-7/LMPG complex up to mixing times of 62 ms. Only at very long mixing times (i.e. 152 ms, Suppl. Figure S7) DHPC-7-protein NOE cross peaks appear for residues 23, 30, 31, 36, 58, 60-62, 64, 65, 94-96, 98, 109, 110, 112, and 114 (Suppl. Figure S7) showing mostly (except Ala34) similar normalized intensities as the corresponding NOEs of YidH in 1H-DPC micelles at a mixing time of 62 ms. No LMPGprotein NOEs are observed probably due to the low concentration of LMPG or due to a larger degree of mobility of LMPG when compared with DHPC-7. For the other residues, still no significant detergent-protein NOEs are observed. A plausible explanation for this pronounced difference in detergent-protein NOEs is that the two membrane mimetics undergo considerable different local dynamics, yielding a distinct intermolecular order parameter S2 (equation 8). Alternatively, the population of the detergent bound to the 32


Page 33 of 50

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protein exemplified by the variable pb in equation 8 may vary (see also Supplementary Material) or the exchange rates of the detergent molecules could be different and would not be in the so-called fast regime (see also Supplementary Material). The first possibility is favored since it is unlikely that the hydrophobic interface of the membrane protein with the detergent is covered significantly different by variable detergents and since it is believed that the detergent molecules are in fast exchange. Following this hypothesis, the detergent molecules in the YidH-DHPC-7/LMPG detergent complex show a major faster local dynamics (i.e. approximately a factor of two) than the detergent molecules in the YidH-DPC complex. It is intriguing to speculate whether the faster presumed dynamics of the DHPC-7 molecules when compared to the 1H-DPC sample could be attributed to the length-difference of the two detergent molecules, which is almost a factor of two (i.e. seven instead of twelve methylenes).

Membrane proteins in detergent micelles are often highly dynamic systems In the case of the membrane-attached HAfp23, the predicted helical wheel pattern of the DPC-protein NOEs was observed experimentally supporting the presence of a tightly packed 3D structure in the system studied as also evidenced by the large number of long range NOEs and well dispersed chemical shifts. In contrast, the smooth pattern of detergent-protein NOE intensities along the helices of HAfp20 and the transmembrane helices of YidH indicate the presence of considerable dynamics of the helices of these membrane proteins. The nature of the dynamics might be either helix rotation or partly unfolding of the tertiary structure increasing the contact surface of a transmembrane or a membrane-attached helix with the membrane mimetics. Further support for this finding in the case of HAfp20 is based on detailed structural studies thereof (88). Additional 33



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Page 34 of 50

support of such (large-scale) motion in the case of YidH is the small chemical shift dispersion (data not shown) of (most of) the chemical shift resonances of the protein including the amide as well as the methyl moieties, which may get averaged upon the dynamics mentioned. Furthermore, for YidH there are only a sparse number of interhelical NOEs and if present they are very weak (data not shown), which may be rationalized by the rotation of the helices or partly unfolding of the tertiary structure. The presence of such large-scale dynamics is also supported by Electron Paramagnetic Resonance (EPR) spectroscopy studies of YidH (Enrica Bordignon, C. T., R. R. personal communication). It is the authors’ notion that for most of the helical membrane protein-detergent complexes studied by solution state NMR there is a low number of interhelical NOEs along with a low chemical shift dispersion, indicating that these proteins are not well structured under the experimental conditions. The absence of a helical wheel pattern of detergent-protein NOEs as exemplified here for YidH and the variant HAfp20 further supports the absence of well folded rigid structures in the studied systems. Whether the suggested finding of extended dynamics of helical membrane protein detergent complexes (such as for YidH) must be attributed to an insufficient membrane mimicry of the detergent used and therefore may be an artifact of the selected conditions, or may reflect the nature of membrane proteins being a loose bundle of stable secondary structural elements that may induce (eventually by a trigger) tertiary structure if required (11) remains to be determined. However, the hypothesis that dynamics is due to insufficient membrane mimicry finds support from comparative studies by EPR between membrane proteins embedded in detergents or liposomes (e.g. (99)).

34


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Conclusions The theoretical considerations on membrane protein detergent complexes using a model composed of several α-helices surrounded by detergent protons supports the notion that detergent – 1HN protein NOE cross-relaxation rates along an α-helix of a membrane protein follow an alteration of segments with strong and weak rates reflecting the helicalwheel character of the helix. This pattern is smoothened if the helix is mainly exposed to detergent/lipid molecules due to either the helix composition of the 3D structure of the membrane protein or dynamics. The character of the dynamics may include for example helical rotation or opening between secondary structure elements. Since these theoretical findings are supported by experimental data, it is suggested that by a simple analysis of the NOE cross peak intensities and the corresponding diagonal, the normalized detergent-protein NOE intensity pattern along a TM helix does not only support the nature of the helix to be a TM or membrane-attached helix but also indicates the presence of tertiary structure such as helix-helix packing, large scale dynamics of the protein helices or/and local protein unfolding - the latter for example attributed to the not ideal membrane mimicry of the detergent. Because of the usually poor experimental information content available for helical membrane proteins this simple analysis is thus regarded as an important aid for NMR structure determination of helical integral membrane proteins.

35



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Page 36 of 50

Acknowledgement This work was supported by a grant from the Schweizerische Nationalfonds (SNF), an ETH internal grant and support from the Department of Chemistry at the University of Illinois at Chicago. We thank Dr. Dave Savage and Dr. Robert Stroud, University of California, San Francisco (UCSF), for providing us the YidH-LIC clone.

Supporting Information Description Description of lipid-protein NOE calculations. Figures showing detergent/lipid – protein NOE build-up curves versus time under chemical exchange, lipid/detergent-protein NOE calculations

under

various

exchange

models,

NOE

build-up

rates

between

detergent/lipid – protein spins in presence of an exchange rate, a simple poly-Ala α-helix model surrounded by detergent/lipid molecules,

15

N relaxation measurements of YidH in

DPC micelles, detergent-protein NOE build-ups and their fits for YidH in DPC micelles, comparison of normalized detergent-protein NOE intensities between the -CH2-groups of DPC or DHPC-7/LMPG mixture and the backbone 1HN of YidH, and saturation transfer experiment results of HAfp23 and YidH in DPC micelles. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1)

Arora, A.; Abildgaard, F.; Bushweller, J. H.; Tamm, L. K. Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Mol. Biol. 2001, 8, 334-338.

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(2)


Almeida, F. C.; Opella, S. J. fd coat protein structure in membrane environments: structural dynamics of the loop between the hydrophobic trans-membrane helix and the amphipathic in-plane helix. J. Mol. Biol. 1997, 270, 481-495.

(3)

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