University of Groningen
Single-molecule fret study on structural dynamics of membrane proteins Aminian Jazi, Atieh
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10.33612/diss.135802718
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Chapter 1:
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Introduction & Background
Atieh Aminian Jazi, Christine Ziegler & Thorben Cordes(unpublished)
Integral membrane proteins perform a range of key functions that are crucial to cells such as material and information exchange with the environment, energy generation and transformation, and cell communication and signalling [4]. Membrane transport proteins, the biological focus of this thesis, mediate the solute transport across the membrane bilayer. Some have roles in the uptake of nutrients and other essential molecules [2], the generation and maintenance of ion gradients [3,12], the extrusion of waste metabolites and toxins [5,4], and the recapture of neurotransmitters and many other substances [2,6].
Given the importance of membrane proteins in so many biological processes, often occurring at the cell surface, it is no surprise that when these processes fail, diseases result [7]. Consequently, membrane proteins constitute the prime target for approximately 60% of all available pharmaceutical drugs [2]. An important step in designing novel therapeutic strategies is thus a detailed understanding of their molecular mechanisms. Structural and functional studies of transporters, aimed at understanding their molecular mechanisms, requires a combined understanding of (static) protein structure and structural dynamics something that I aspire here for active transporters. In this thesis, l will describe my efforts to establish novel methods to allow the single-molecule investigation of structural heterogeneity and dynamics of substrate-binding domains of ATP-binding cassette transporters [9,12] TRAP-transporters [11]and the secondary transporter BetP from Corynebacterium glutamicum [1,13].
The thesis contains the following chapters: Chapter 1 provides the scientific background on membrane transporters, the model protein BetP, all biophysical methodology and a synopsis of all scientific parts. In Chapter 2, I establish novel labelling schemes and advanced fluorop hores for membrane proteins. In Chapter 3, I overcome the hurdles to label BetP, as a trimeric protein with labels for single-pair FRET studies. In Chapter 4, I use the established methodologies to elucidate the activation mechanism of BetP, i.e. the conformational changes of the regulatory
C-8
terminal domain upon activation by the non-physiological stimulus K+. Chapter 5 summarizes and
discusses all results in light of the current state-of-the-art.
1.1 Background on membrane transporters
Molecules such as ions, peptides, small molecules, lipids and macromolecules and others are not only passively transported via channels or pores down their concentration gradient but can also be actively transported against chemical gradients. Membrane transporters are classified into channels or carriers (transporters) based on their biochemical functions, structure and location within the membrane. Transporter function by the alternating access model of transport in which, the substrate binding site can only and specifically be accessed from one side of the membrane at a time [14,13] (Figure 1.1). Many integral transmembrane proteins span the lipid-bilayer via stabilizing hydrophobic interactions [38]. Integral membrane transporters exhibit high specificity for their substrates. In contrast peripheral membrane protein are able to dissociate from the membrane to participate in a variety of cellular interactions such as lipid transport and cell signalling. Peripheral membrane proteins and their interactions with the lipid -bilayer are stabilized by both electrostatic or hydrogen bonds or other non-covalent interactions between the polar heads and polar regions of the protein [13,14].
Figure 1.1| Functional classification of membrane transport proteins: Cartoon sketch of different membrane proteins that passively or actively drive transport of substrates across membranes: (a) channels/pores that facilitate passive diffusion, (b) primary-active transporter where conformational changes in a transport protein
are driven by, e.g., ATP-hydrolysis and (c) secondary-active transporters that use an electrochemical gradient to co-transport a substrate. Substrate is shown as a black dot (a/b/c) and the co-substrate as a smaller red dot (c).
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Active transport requires chemical energy stored in electrochemical gradients of ions (co-substrate) or molecules such as ATP to move the main substrate across the membrane. Primary-active transporter utilize the energy provided by ATP hydrolysis, for example in ABC transporters [10], to allow alternating access of a ligand binding site in the transmembrane domains. Secondary-active transporters mediate the uphill transport of the substrate by coupling it to the downhill flow of a co-substrate, often an ion (for example a proton or sodium-ion) in a thermodynamically unfavourable direction across the cell membrane, see Figure 1.1. The transporter investigated here is BetP [8,20] which is a member of the BCCT family of secondary transporters (betaine-choline-carnitine-transporters). These secondary transporters mediate the transport of one substrate, mostly a substrate with a trimethylammonium group against a concentration gradient using co-transport or counter-transport of another substrate or an ion. Members of the BCCT family are found in Gram-positive bacteria (BetL from Listeria monocytogenes [12,13]), Gram-negative bacteria (e.g. OpuD from Vibrio cholerae [43]) but also in archaea (e.g. glycine betaine transporter from Methanogenium cariaci [2,26]).
1.2 Scientific background on sodium-symporter BetP
BetP from Corynebacterium glutamicum is a well-characterized member of the BCCT family, Transporters belonging to the BCCTs family share common functional and structural features related to trimethylammonium substrate specificity [13], transported substrates contain a quaternary ammonium group. BetP’s function is transport of betaine via a sodium-symport mechanism. Crystal structures reveal that BetP is an asymmetric trimer [21]. Moreover, they show that a conserved tryptophan motif, the signature motif of the BCCT family, forms the binding site for the quaternary ammonium [15]. The transport core consists of two tightly nested structurally inverted repeats of five transmembrane helices allowing occlusion of substrate during transport [21].
Crystal structures demonstrated that BetP is an asymmetric trimer (Figure 1.2A), in which each of three protomers can independently adopt distinct conformations for transport. These were assigned as individual transport states in the alternating access cycle that allow uphill substrate
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transport driven by the electrochemical Na+ potential leading to accumulation of glycine betaine,
which is the exclusive substrate for BetP, to molar amounts in the cytosol under hyperosmotic conditions [13]. BetP requires both hyperosmotic stress as well as an elevated internal K+
concentration for full activation. However, as K+ is always above the Kd of BetP (220mM) in E.
coli phospholipid liposomes , the K+ activation observed in proteoliposomes is considered as a
non-physiological stimulus [1,23]. Anyway, the detailed mechanism of stress sensing and activation is not yet described on atomistic level, but multiple functional and biochemical data have identified the C-terminal domain being key for sensing and regulation in BetP (Figure 1.2B). Thus, BetP has two distinct functions: sensing of osmotic stress and regulated transport of glycine betaine. It is assumed that the 45 amino-acid long C-terminal helix domain of BetP binds cytoplasmic K+,which is a pre-requisite to activate BetP during hyperosmotic stress [21,25]. The
catalytic domain of BetP consists of 12 transmembrane helices (TM) and is divided into a transporter core of two inverted five-helix-repeats (TM3-TM12) and the two N-terminal helices TM1-2, which contribute to the trimer contacts. The symmetry between two repeats (TM3-TM7 and TM8-TM12) is a key to the alternating access mechanism in BetP [39]. Due to the variety of structural and biochemical studies, BetP serves as a paradigm for the mechanism osmoregulated transporters [28].
Figure 1.2| Structural and functional characteristics of BetP sodium-symporter from the BCCT family. Structure and FRET properties of the homotrimeric C252T/S516C BetP mutant. (A) Side and top views of the crystal structure of the mutant marking the three label positions and related distances. Protein Data Bank entry 4AIN. (B)
Normalized uptake rate of Cys-less BetP (wt) and BetP cysteine mutant C252T/S516C in E. coli cells depending on osmotic stress. The relative rate of uptake of 14C-labeled betaine for wild-type protein (green) is comparable to that of the mutant protein (red, C252T/S516C), which exhibits one-third of the total wild-type activity.
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The 45 amino-acid long positively charged C-terminal domain (Figure 1.3, positively-charged residues in C-terminal domain are shown in blue) of BetP is suggested to sense directly hyperosmotic stress, however the stimulus that it is perceived by this domain is not known. As activation of BetP when reconstituted into proteoliposomes can be mimicked by increasing cytoplasmic K+ concentration, the non-physiological K+ stimulus was used in the past to
investigate mechanistic features of activation, e.g. in spectroscopic measurements using AFM or PELDOR [23,28]. The biochemical properties of C-terminal domain has been studied extensively during the past decades and revealed that the osmodependent regulation of BetP is strongly dependent on the surrounding biochemical environment of the helices [28]. The osmoregulatory function was seen in other transporters such as OpuA, a type I ABC importer [33]. Specific activation by potassium ions via a cytoplasmic C-terminal domain and instant regulation of transport activity was, however, only observed in BetP transporter [39].
Figure 1.3| BetP trimer in its surface representation. Positively-charged residues are color-coded in blue with possible label positions for fluorophores shown in orange. The blue colour positions indicate possible interaction within the C-terminals of adjacent protomers within BetP protein, PDB: 4C7R.
In order to study the activation mechanism of BetP in more detail, this thesis establishes smFRET experiments on BetP with the goal to study the conformational states of the C-terminal helix. For this purpose, we employed Förster-resonance energy transfer (FRET), a nanoscopic distance
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ruler, in combination with single-molecule detection to investigate the conformational states and dynamics of BetP. smFRET provides a spatial resolution of sub-nanometers with a dynamic range of 2-10 nm with sub-second temporal resolution [40]. This combination is ideal to understand the structural changes required for osmoregulation in BetP and to characterize the flexible structural part within the C-terminal domain.
From a biological viewpoint I mainly investigate the effect of potassium-induced activation and conformational switching of the BetP C-terminal domain using smFRET in combination with new crystal structures which were co-crystallized with K+ and Rb+. For this, we designed various BetP
cysteine-mutants based on a cysteine-less BetP (C252T, TM5) containing engineered cysteines at the periplasmic and cytoplasmic positions in transmembrane helices and C-terminal domain of BetP. One example position of a potential periplasmic residue (S516C) is shown in Figure 1.2A.
Besides the achievement to label BetP for smFRET studies another major adv ance in my work was the use of amphipols (Figure 1.4) to mimic the natural membrane environment. Amphipols are amphipathic polymers that were successfully used before in order to investigate the structure of membrane proteins in structural biology and spectroscopic studies, e.g., biochemical characterization of transporters or for cryo-EM [22,37]. In this study, we used Amphipol polymer (A8-35) to stabilize the BetP variants for characterization and biophysical experiments of BetP in a detergent free solution [27,22].
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Figure 1.4| Chemical Structure of Amphipol (A8-35), polyacrylate-based APol. Amphipathic polymer that allows solubilisation of membrane proteins in a detergent-free aqueous solution [41].
The hydrophilic backbone of these polymers (Figure 1.4) interacts with hydrophobic elemen ts within membrane proteins with high affinity and covers the transmembrane regions and keeps them water soluble in a protein-amphipol complex. Such a strategy is especially useful for smFRET experiments with diffusing molecules in the absence detergent [27]. In chapter 4 of this thesis, we used Amphipol (A8-35) for the first time in smFRET studies of C-terminal domain in Amphipol-stabilized variants of BetP.
1.3 Scientific background on biophysical methods and smFRET
Förster Resonance Energy Transfer (FRET) has become a powerful method to probe the structural dynamics and conformational heterogeneity of biomolecules [1]. FRET is a fluorescent -based photophysical mechanism where excitation energy is transferred from an excited donor fluorophore (D) to an acceptor fluorophore (A) in close-proximity via non-radiative dipole-dipole coupling; Figure 1.5A [18,30]. Due to its steep distance dependence FRET can be used as a spectroscopic ruler with a dynamic range of 3-8 nm (Figure 1.5B).
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Figure 1.5| The concept of FRET. A) An electromagnetic transmitter-receiver is a macroscopic analog for B) the molecular dipole-dipole coulombic interaction between donor, D, and acceptor, A, fluorophores. The dependence of the efficiency of energy transfer from D to A on their distance provides a molecular ruler with a high dynamic range on the 2-8 nm scale. C) Simplified Jablonski diagram of donor D in the presence of a FRET acceptor A. After excitation (kex) to the excited competing pathways deplete the excited state S1: (i) kD, T which is the sum of radiative
and non-radiative decay rates from S1 to S0. Radiative decay results in fluorescence emission; (ii) Förster-type
energy transfer kFRET to an acceptor fluorophore A decaying with kA. Figure panel A/B and corresponding caption
were reprinted with permission from Lerner & Cordes et al., Science 2018. Figure panel C and corresponding caption was reprinted with permission from Ploetz & Lerner et al., Scientific Reports 2016.
FRET can be described using a Jablonski diagram that explains the occurrence of FRET, i.e., excitation energy transfer as an additional pathway for donor excited state deactivation in a non-radiative fashion. The transfer of excitation energy occurs from the excited donor fluorophore to the nearby acceptor through a non-radiative dipole-dipole interactions (Figure 1.5C) [18,34]. Ideally, the FRET efficiency E depends only on the distance between both donor and acceptor fluorophores providing the ruler character as shown in Figure 1.5B. This holds only for the case that the relative orientation of the donor-emission and acceptor-absorption transition-dipole moment, spectral parameters (donor-emission spectrum, acceptor absorption spectrum) [36,40] are constant whenever conformational changes occur. In this case the FRET efficiency can be calculated via ratio metric measurements of fluorescence intensity ratios or fluorescence lifetimes (eqn. 1):
Eqn. 1.
In eqn. 1 FDA is the acceptor-fluorescence intensity after donor excitation and FDD the
donor-fluorescence intensity after donor excitation corrected for spectral cross-talk and quantum yield differences. DA is the fluorescence lifetime of the donor-acceptor (DA) species, D the
fluorescence lifetime of the respective donor-only (D-only) species. R is the distance between donor and acceptor, and R0 marks a dye-pair specific separation where the FRET transfers
efficiency is equal to 50% (see Figure 1.5B), For accurate calculation of R-values, knowledge of the Förster radius R0 and determination of accurate FRET values E are required [18,29].
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FRET is a powerful technique to monitor both inter- and intramolecular interactions that induce changes in donor-acceptor distances when being present on biomolecules. Such applications include protein-protein interactions, estimation of intracellular ion concentrations, kinetic studies for analysis of the enzymatic activities and protein conformational changes [18,29]. Figure 1.6 shows the main method used in this thesis.
Figure 1.6| Dynamic structural biology using smFRET. A) Scheme of data of microscopy setup for µs-ALEX measurements using a confocal microscope. The experimental setup is a combination of single -molecule fluorescence microscopy and spectroscopy which can be used to determine conformational states or dynamics in solution including conformational heterogeniety. Figure panel B) and caption were reprinted with permission from Lerner & Cordes et al., Science 2018.
For my work, single-molecule detection via confocal microscopy was combined with Förster-resonance energy transfer (FRET) via ratiometric detection of donor- and acceptor fluorescence [35,42]. The setup was able to excite both donor- and acceptor fluorophore to produce fluorescent bursts whenever labelled proteins would diffuse through the confocal excitation/detection spot (Figure 1.6A, top and Figure 1.6B). This information was used to extract information on the existing conformational states of BetP and other proteins in solution and possible conformational dynamics.
1.4 Thesis outline
The thesis contains the following chapters with novel scientific contributions: In Chapter 2, I establish novel labelling schemes and advanced fluorophores for membrane proteins. In Chapter
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3, I overcome the hurdles to label BetP, as a trimeric protein with labels for single-pair FRET studies. In Chapter 4, I use the established methodologies to elucidate the activation mechanism of BetP, i.e., the conformational state of the regulatory C-terminal domain, relation conformational dynamics to static structure snapshots from X-ray crystallography.
Fluorescence labelling of membrane proteins and isolated soluble domains (chapter 2) :
Site-specific labelling of proteins and biomolecular complexes is a fundamental prerequisite for Förster-resonance energy transfer (FRET) experiments. Yet, especially for hydrophobic membrane proteins, only few approaches yielding high donor-acceptor fractions and favourable photophysical properties are established. In this chapter I provide novel methods for labelling (membrane) proteins with green and red organic fluorophores. Methods with resin-immobilized proteins (substrate-binding proteins) and free-diffusing membrane proteins (BetP/BasC) are compared and evaluated. Finally, I present smFRET characterizations of the performance of fluorophore-dye conjugates (see Figure 1.7) on substrate-binding domain 2 of ABC importer GlnPQ as a model system.
Figure 1.7| Design concept for photostabilizer–dye conjugates. UAAs are used to combine an organic fluorophore covalently with a photostabilizer on a biomolecular target or linker structure. This strategy was used to improve the properties of fluorescent dyes on isolated membrane transporter domains.
Caged FRET (chapter 3): Förster-resonance energy transfer (FRET), a nanoscopic distance ruler,
in combination with single-molecule detection has become a powerful tool to investigate the structural dynamics of biomolecular systems [35,8]. Here in the third chapter of this thesis, we introduce and elaborate a novel single-molecule approach to overcome the problems arise when using regular smFRET technique to characterize a complex biochemical species, such as
multi-17
labeled DNA and trimeric BetP. FRET labelling of a multi-subunit complex leads to the generation of heterogeneous mixture of FRET species that hamper the precise smFRET data acquisition (e.g., donor-acceptor-acceptor, Figure 1.2).
Figure 1.8| Working principle of Cage FRET methodology with corresponding time traces. Cartoon view of the excitation volume where diffusing species produce only green signals (top left, caged acceptor) and both green and red signals (top right, UV-activated acceptor) with corresponding smFRET photon streams of caged (left bottom) and after UV activation process (right bottom).
In this work, we establish caging of cyanine fluorophores and caged rhodamine dyes as photoactivatable fluorophore, i.e., chemical deactivation fluorescence, for single-molecule FRET experiments of freely diffusing molecules. The novel ‘caged FRET’ method was used to investigate the structure of the homotrimeric membrane transporter BetP, as an example of a multi-subunit protein, and also nucleic acids containing more than two fluorescent labels. The obtained results revealed that chemical caging and photoactivation (“uncaging”) by UV light allows temporal uncoupling of convoluted fluorescence signals from multiple donor or acceptor molecules. Therefore, the recovered fluoresce signals are use d to extract the desired FRET-related information [8]. Our study suggests that ‘caged FRET’ technique is not only suitable for smFRET analysis of complex biochemical systems, but it may also be used to study the intermolecular details of low-affinity binding interactions with diffusion-based smFRET. I will focus on Caged FRET methodology to elucidate the structure of membrane transporter BetP on chapter 3 of this thesis.
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smFRET studies of BetP (chapter 4): This chapter was dedicated to provide new information on
the “molecular switch model” of BetP regulation, which describes how the C-terminal domain of BetP is changing conformation in order to detaches itself from the membrane in order to up -regulate transport. While the alternating access mechanism of betaine-sodium coupling and transport is very well described from a structural perspective, the conformational switching of the C-terminal domain during activation is not known on molecular level. This is attributed to the fact that crystal contacts force the C-terminal domain always in one distinct conformation, in which only one out of three terminal domains are resolved entirely. Here, we assessed potassium-induced conformational changes of the C-terminal domain by employing Förster-resonance energy transfer (FRET) in combination with single-molecule detection – an approach that allowed us to elucidate the conformational changes of the entire -helical C-terminal domain by using different labelling positions. For this, we first developed a direct approach to characterize the structural arrangement of BetP in different biochemical environments with homotrimeric cysteine mutants in the C-terminal helix (Figure 1.9A). We observed major differences in the conformational states in polymeric amphipols compared to detergent environment (Figure 1.9B). The observed differences and specifically the conformational space of the C-terminal domain identified in amphipol likely reflects a more native membrane-like state than detergent. Finally, we investigated the effect of potassium on the conformational states of the C-terminal domain. We demonstrate that in a negatively charged membrane -mimic conformational distribution of the C-terminal helices is narrowed down in response to increasing the concentration of K+ (Figure 1.9B). We assume that crystallization contacts populate one of
these conformations. From a new Rb+ co-crystallized BetP structure and in the light of our new FRET data we could deduce that one purpose of K+ binding is to strengthen the interaction of a
Cterminal domain with the cytoplasmatic loop of the adjacent protomer by weakening lipid protein interactions. However, again based on our FRET data (which shows protein is in a pre -activated state when it is in a non-ionic detergent) can now attribute the conformation observed in the X-ray crystal structures of BetP to a pre-activated state, in which these lipid interactions are not present as the protein is in a non-ionic detergent. Our studies also show that neither sodium nor lithium have a comparable effect on the conformational state of the protein. Our
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findings confirm the role of potassium as a pre-requisite for full-activation to counteract the strong lipid-protein interaction in C. glutamicum, membranes consisting of negatively charged lipids.
Figure 1.9| smFRET studies of BetP (584C homotrimer) using ALEX spectroscopy. A) 2D-ALEX histogram showing three distinct labelling species in E*/S plot, where only the central DA population was selected for further analysis. B) 1D FRET efficiency plots of BetP in DDM (detergent) with no potassium versus BetP in amphipol (AMP) at different concentrations of potassium, showing that the activated state of the protein is formed both at high potassium concentrations in the native-like amphipol-state and in DDM without potassium.
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