interaction partners
Xu, X.
Citation
Xu, X. (2009, January 21). Transient interactions studied by NMR : iron sulfur proteins and
their interaction partners. Leiden. Retrieved from https://hdl.handle.net/1887/13428
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13428
General Introduction
Protein-protein interaction
Protein-protein interaction plays important roles in most cellular processes, such as signal transduction, transcription regulation, cell cycle, and immune recognition (1). Understanding of these interactions can provide the insight in the molecular mechanisms of disease and lead to the design of new therapeutic approaches (2).
Some proteins interact for a prolonged time to form a stable complex. Others only interact briefly and dissociate quickly. The affinity of a protein-protein interaction can be described by the equilibrium dissociation constant (Kd), which is equal to koff/kon: koff and kon are the rate constants of dissociation and association reaction, respectively. Kd observed in biological protein-protein interactions ranges from 10-2 to at least 10-16 M (3,4). The affinity and life time of a protein complex are well correlated with its function in cellular processes. For protein complexes formed by enzymes and inhibitors, such as barnase-barstar complex (5), a tight binding is required to ensure that the enzyme activity is completely switched off by binding to the inhibitor. In some other complexes, the function of complex formation is to relay cellular signals. This requires that one protein interacts with multiple partners. These interactions are often transient. Electron transfer complexes formed by some redox proteins are of great interest because they are often very transient, with lifetimes sometimes less than 1 ms. Characteristics of electron transfer complexes includes both high association (kon, 107-109 M-1s-1) and dissociation rates (koff ≥ 1000 s-1), with Kd in the range of μM-mM (6). High association rates facilitate the complex formation while high dissociation rates ensure fast turn-over of these complexes.
The interesting question to ask about these electron transfer complexes is how the balance between the specificity and low affinity is achieved.
The two-step model of protein-protein complex formation
Protein-protein association rates (kon) span many orders of magnitude (103-1010 M-1s-1). In a diffusion-limited association, the rate value in the range of 105-106 M-1s-1 can be predicted based on the Smoluchowski equation (7) combined with additional stereo-specific alignment constraints (8). However, for some protein complexes, the observed associated rates are as high as 109 M-1s-1. The concept of encounter complex and electrostatic steering were developed to account for this discrepancy. In the two-step model of complex formation, the encounter complex is defined as a loosely-bound intermediate state before the formation of the final, well-defined complex. Two proteins first diffuse randomly until they sense the electrostatic field of the other. With the electrostatic steering, two proteins can form an encounter state which can be described as an ensemble of multiple iso-energetic orientations of protein complexes. In this model, the role of the encounter complex is to accelerate complex formation by a reduced- dimensional search (9). The long-range electrostatic force is a major driving force of encounter complex formation (5,10). Most often, the encounter state is rarely observed experimentally because it exists only briefly and quickly converts into the final well-defined, productive complex. However, a recent study on electron transfer complexes suggests the population of the counter state in those complexes varies (6). Some electron transfer complexes between proteins with non-physiological origins can even exist mainly in an encounter state.
Characterization of this kind of complex will advance our understanding of the two-step model of protein-protein interaction.
Fast dissociation of transient complexes
Contrary to the stable complex formed between proteins in immune recognition or enzyme inhibition, in which fast dissociation must be avoided, transient
complexes formed by electron transfer proteins often require a high turnover rate. The high turn over rate can be achieved by high dissociation rate constants (koff).
The fast dissociation of transient complexes can be controlled by several factors.
Surface complementarity of two proteins in the complex is not an essential condition for a transient complex formation (11). Lack of surface complementarities also lead to loose packing of interface of complex. For some electron transfer complexes, the balance of association and fast dissociation is well achieved, through fine-tuning the population of encounter state and the well-defined state. Large conformational changes are usually not observed for proteins in these complexes.
For some electron transfer protein complexes, another mechanism is proposed to account for the turn-over of the complexes. The redox-state dependent changes of the proteins or subtle conformational change can function as a switch in regulation the affinity of two proteins after the electron transfer (12).
Proteins and complexes studied
Iron sulfur proteins
Iron-sulfur clusters are ubiquitous prosthetic groups that are required for various fundamental life processes (13). Iron-sulfur proteins are proteins characterized by the presence of iron-sulfur clusters containing sulfide-linked di-, tri-, and titra- iron centers in several oxidation states. It was not until around 1960 that in photosynthetic studies on various organisms, evidence accumulated for the existence of iron-containing non-heme proteins. Later, by the mid-1960s, these proteins were shown to contain complexes of iron and cysteinate sulfur atoms and the inorganic sulfide (14). Iron sulfur proteins are found in photosynthetic and respiratory electron transport chains. Iron sulfur clusters are suited for their primary role in mediating electron transfer due to the ability to delocalize
electron density over both Fe and S atoms. Iron-sulfur clusters are involved in electron transfer contain 2Fe-2S, 3Fe4S, 4Fe-4S, or 8Fe7S core units, with cysteinate (in most of cases) tetrahedrally coordinated at Fe site. In addition to their primary role as an electron transfer mediator, FeS clusters can constitute active sites of a wide range of enzymes.
Ferredoxins (Fd) are a group of iron-sulfur proteins comprising more than one Fe and labile sulfur and exclusively displaying electron transfer activity but not catalytic function. Extensive research on Fds revealed that the 2Fe-2S Fds constitute a superfamily in which the proteins have one 2Fe-2S cluster per molecule and similar peptide chain folding. Three subfamilies can be further defined, including halophilic archaebacterial Fds, vertebrate Fds (adrenodoxins) and plant-type Fds.
Adrenodoxin
Adrenodoxin was first discovered and isolated as a brown-colored non-heme iron protein from bovine adrenal cortex mitochondria in 1965 (15). Later, it was further characterized as a negatively charged 14.4 kDa iron-sulfur protein that belongs to the broad family of the 2Fe-2S type Fds found in plants, animals and bacteria. It is involved in steroid hormone biosynthesis of vertebrates. The mammalian mitochondrial steroid hydroxylating system of the adrenal gland consists of three components, a NADPH dependent adrenodoxin reductase (AdR), Adx, and a cytochrome P450 (P450scc). In this system, Adx functions as a soluble electron carrier between AdR and several cytochromes P450, including P450scc which catalyses the side chain cleavage of cholesterol (16).
The X-ray structures of truncated form (4-108) (17) (Figure 1A) and wild type of bovine Adx (18) were determined in 1998 and 1999, respectively (PDB entries 1ayf and 1cjc). The truncated Adx exists as monomer whereas the full length form is a functional dimer. The unstructured C-terminal peptide was suggested to be the dimerization region (19,20). Adx comprises two domains, named the core
and recognition domains. The core domain includes the FeS cluster and residues 5-55 and 91-108, while the recognition domain contains the key residues for the interaction of Adx with AdR and cytochromes P450.
Cytochrome c
Cytochrome c (Cc) was first discovered and isolated as a principal component of the eukaryotic respiratory chain in 1920s (21,22). Later, Cc was found across a spectrum of species including plant, animal, and unicellular organisms. The primary sequence of Cc consists of around 100 amino acids. The comparison of Cc structure from more than 100 species suggested this protein is highly conserved.
Yeast mitochondria Cc is positively charged and highly soluble in solution. It contains heme as a prosthetic group. In c-type cytochromes, the heme group is covalently linked to the polypeptide via two thioether bonds to two cysteine Figure 1.1. Structure of bovine adrenodoxin 4-108 (A) and yeast iso-1 cytochrome c (B). A. Ribbon representation of crystal structure of bovine Adx 4-108 (PDB entry, 1AYF), core domain is in green and recognition domain is in blue; the iron sulfur cluster is shown as red spheres. B. Ribbon representation of the crystal structure of yeast Cc (PDB entry 1YCC), the heme group is shown in red sticks.
residues of Cis-X-X-Cis-His motif, where His is one of the axial ligands. The heme contains an iron which can have two oxidation states, FeII and FeIII. The reduced form is diamagnetic and oxidized form paramagnetic.
Both high resolution X-ray and NMR structures of ferrous (23,24) and ferric Cc
(25,26,27) have been reported. The structure fold of Cc is highly conserved and consists of five α-helices and a short β-strand (Figure 1.1 B).
The complex of Adx and Cc
In vitro, the electron flow from AdR to Adx can be monitored by the following electron transfer from Adx to some non-physiological electron acceptors such as K3Fe(CN)6 or mitochondrial cytochrome c (Cc), because the electron transfer from AdR to Adx is the rate-limiting step (28). The observation that fast electron transfer occurs between two proteins Adx and Cc suggested a non-physiological complex is formed. Recently, the interaction of bovine Adx and yeast Cc was investigated by solution NMR spectroscopy (29). Complex formation of two proteins at low salt conditions was suggested from the line broadening and chemical shift change in two-dimensional NMR titrations. The binding association constant was determined to be 4×104 M-1. From the chemical shift mapping, it was suggested that Cc may use the heme edge region to sample a large surface of Adx to form a dynamic ensemble.
Plant-type ferredoxin
The first plant-type ferredoxin Fd was discovered in spinach chloroplast (30), and later it was found present not only in the stroma of chloroplast of higher plants and eurokaroytic algae but also in the cytoplasm of cyanobacteria.
The primary function of plant-type Fd is to deliver electrons from photosystem I to ferredoxin:NADP oxidoreductase (FNR). This enzyme catalyzes the formation of NADPH, an essential component of the carbon fixation process. In addition, plant-type Fd can also serve as electron donor to other cellular
enzymes, including nitrite reductase involved in the reduction of NO2 to NH4+, glutamate synthase in the synthesis of glutamic acid from glutamine and 2-oxo- glutarate, sulfite reductase for the conversion of SO32- to S2-, and ferredoxin:thioredoxin reductase, which functions as a central protein in regulation of the activities of various enzymes in carbon metabolism (31).
Plant-type Fds are highly soluble and rich in acidic residues. Most of plant-type Fds consist of 95-98 amino acid residues. It has a motif of CX4CX2CXnC which includes four cysteines ligands for the 2Fe-2S cluster. Electron paramagnetic resonance (EPR) spectroscopy showed that the two iron atoms in the cluster in oxidized state are antiferromagnetically coupled to give no net spin at low temperature. Upon reduction, one iron is reduced to give the new spin S=1/2.
The first crystal structure of the plant-type Fd from cyanabacterium was determined in 1980 (32). Three-dimensional structures of plant-type Fds solved by X-ray crystallography and NMR exhibit extensive similarities. Figure 1.2 shows the crystal structure of Fd from Synechocystis sp. PCC6803. The protein has a typical β-grasp fold, including a core of a 4-stranded β-sheet and one α-helix flanking the β-sheet. The core is surrounded by the iron-sulfur binding loop and
Figure 1.2 Ribbon representation of the crystal structure of Fd from Synechocystis PCC 6803 (1OFF).
The 2Fe-2S cluster is shown in spheres.
the segment containing the second α-helix. The 2Fe-2S is located close to one surface whereas the β-sheet forms the other.
Ferredoxin:thioredoxin reductase
In 1960s, the search for ferredoxin-linked carboxylation reactions in chloroplast led to the unexpected observation that the reduced ferredoxin stimulates the CO2 fixation in the presence of fructose 1,6-bisphophatase, an enzyme of Calvin cycle. Later, ferredoxin/thioredoxin reductase, first named ‘assimilation regulatory protein A’, was found to be essential for this stimulation. This protein was first co-purified with another “assimilation regulatory protein B’ which is thioredoxin (Trx) (33). Since then it became clear that these three proteins functioned in what is now known as the ferredoxin/thioredoxin system, a reductive mechanism that links light to the regulation of enzymes involved in photosynthetic carbon assimilation (34).
FTR was found in oxygenic photosynthetic organisms (the prokaryotic cyanobacteria and chloroplast of plant) and in amyloplasts in eukaryotes. The enzyme is different from NADPH-dependent thioredoxin reductases (NTRs) which are flavoproteins and accept electrons from NADPH. FTR is an iron- sulfur protein containing a redox active disulfide bridge that utilizes a 4Fe-4S cluster to mediate the electron transfer from the one-electron carrier ferredoxin to the two-electron acceptor Trx. It is a heterodimer consisting of a 13 kDa conserved catalytic subunit and a variable subunit with difference sizes (range from 8 to 13 kDa) among different species. The N-terminal extension of latter subunits with inconstant length accounts for the size difference of FTRs from prokaryotes and eukaryotes. Removal of part of the N-terminal extension of spinach FTR significantly increases its stability without affecting its catalytic properties (35). The evidence suggests that variable subunit may function as a chaperone.
Fully active recombinant FTR expressed in E. coli has been obtained for the enzyme from spinach and Synechocystis sp PCC 6803. The DNA construct used for expression includes the coding sequences for two subunits separated by a spacer region and a second ribosome binding site. Due to its prokaryotic nature of the gene, the expression of Synechocystis FTR is significantly better than that of spinach FTR. Highly stable Synechocystis FTR proved to be ideal for crystallography studies.
The crystal structure of Synechocystis FTR was determined to 1.6 Å resolution
(36). It is a thin, disk-like molecule of only 10 Å across its center where the iron sulfur cluster is located (Figure 1.3). The heart-shaped variable subunit is an open β-barrel structure with five anti-parallel strands. The catalytic subunit has an overall α-helical structure consisting of five helices with loops inserted between the helices containing iron-sulfur ligands and redox active cysteines.
The cubane 4Fe-4S iron sulfur cluster is located on one side of the catalytic subunit and covered by the redox active disulfide. Solely based on the structure of FTR, the prediction was made that FTR can use the two sides to
Figure 1.3. Crystal structure of Synechocystis FTR. The catalytic and variable subunits are shown in green and purple ribbons, respectively. The 4Fe-4S cluster is shown in orange spheres and the redox active disulfide is shown in red sticks.
simultaneously dock with Fd and Trx. This interaction was confirmed by recent X-ray crystallography studies (36).
The catalytic domain of FTR has seven highly conserved cysteine residues, six of which are organized in two Cys-cisPro-Cys (CPC) and one Cys-His-Cys (CHC) motifs. In the Synechocystis FTR, C57 and C87 form the active site disulfide, the other four cysteines C55, C74, C76 and C85 are the ligands to the 4Fe-4S cluster. UV, visible and CD spectroscopy data, combined with the absence of an EPR signal indicates the that the FeS cluster is 2+ (S=0) for the resting enzyme (37,38). Mössbauer spectroscopy study suggested a weak interaction between the iron sulfur cluster and one disulfide cysteine in the resting state (39). Further insights into the reaction mechanism were also revealed by Mössbauer spectroscopy and confirmed by recent X-ray crystallography studies (39,40). The detailed reaction mechanism will be discussed in chapter 5.
Thioredoxin
Thioredoxin was first purified in 1964 from E. coli as a hydrogen donor for ribonucleotide reductase (41). Since then, the research on thioredoxin leads to surprising discoveries on the various regulatory functions of this protein (42), which include enzyme regulation, response to oxidative stress, transcription and translation.
Thioredoxin is a ubiquitous, small (12 kDa) protein in all living cells and has a characteristic tertiary structure, termed the thioredoxin fold which includes four α-helices surrounding a five-stranded β−sheet (Figure 1.4). Its redox active site is made up of two neighboring cysteines in a conserved WC(G/P)PC motif. In plants, the oxidized form of Trx can be reduced either by the NADPH-linked system in which Trx receives electrons from the NADPH via NTR, or by Fd/FTR system in which Trx receives electrons from Fd via FTR. Unlike bacteria and animals, in plants a large number of genes encode 19 different Trxs which can be grouped into six subfamilies. Chloroplast contains four types m, f ,
x and y whereas o is located in mitochondria and h type is distributed over various compartments (43).
Complexes of Fd/FTR/Trx
In the redox regulation pathway linking light to the activity of associated enzymes of photosynthesis (Figure 1.5), the interaction between Fd, FTR and Trxs plays a central role (44). To transfer electrons from Fd to Trxs and finally to the target enzymes, FTR is able to interact with Fd and Trx simultaneously. The structures of the non-covalent complex Fd/FTR, covalent complexes FTR/Trx and ternary complex of Fd/FTR/Trx-f were determined by X- ray diffraction (40) (Figure
1.6).
Figure 1.4 Ribbon representation of the crystal structure of spinach Trx-m. The redox active site including the disulfide is in red.
The complex between Fd and FTR is formed under low ionic strength. The crystal structural shows that Fd docks to one side of FTR and the complex is stabilized by a number of hydrogen bonds, salt bridges as well as hydrophobic interactions between two proteins. FTR can use the other side for the docking of Trxs (m and f) and the interaction involves a transient inter-disulfide bridge formation between C57 of FTR with C37 of Trx-m (C46 for Trx-f ) (Figure 1.6B). The stabilized complexes formed between FTR and Trx mutants (Trx-f C49S and Trx-m C40S) were crystallized and the structure shows a special five- coordinate 4Fe-4S3+ cluster formation in the complex, which confirmed the reaction mechanism proposed from previous spectroscopy studies.
Figure 1.5. The Fd/Trx system of oxygenic photosynthetic organisms. The light stimulates Fd reduction. Electrons are then transferred to target enzymes via the interaction system including Fd, FTR and Trxs. Adapted from Quarterly Reviews of Biophysics 33, 1 (2000), pp. 67–108, with the permission from Cambridge press.
Figure 1.6. Crystal structures of complexes between Fd, FTR and Trx. The 2Fe-2S and the 4Fe-4S clusters are shown in spheres and sticks, respectively.
A. Binary complex between Fd and FTR (PDB: 2PVG).
B. Cross-linked complex of FTR with Trx m C40S (PDB: 2PUK).
C. The ternary complex formed between Fd and cross-linked FTR/Trx f C49S (PDB: 2PGO).
The ternary complex formation between Fd and cross-linked FTR/Trx was studied by gel filtration (45) and the crystal structure was determined (Figure 1.6 C). But so far, the non-covalent ternary complex has not been crystallized, possibly due to its transient nature.
NMR methods to probe protein-protein interaction
Various genetic, biochemical and biophysical approaches have been developed to detect PPI in vivo and in vitro. Solution NMR techniques have proved to be an indispensable tool for the study of protein interaction and offer invaluable information on the study of protein interaction interface, protein complex structure, kinetics and dynamics of protein-protein interaction.
Chemical shift perturbation and mapping
Interface mapping by NMR chemical shift perturbations is well established and widely used to probe protein-protein interactions (46). A 15N-1H HSQC spectrum is firstly recorded for a free 15N labeled protein and then a series of HSQC spectra is aquired when an unlabeled interacting partner is titrated in. The 15N-
1H HSQC spectrum gives a fingerprint of protein structure and each cross-peak represents an amide group. For the residues in the interface, the interaction causes chemical perturbations which lead to the chemical shift changes of peaks in the spectra. The binding site of the interaction can be delineated by comparison of the spectra of the protein for its free and bound states when the resonance assignments are known. HSQC chemical shift mapping not only provides the information on the binding interface, but also time scale of association and dissociation and the affinity of binding (47). However, chemical shift perturbation fails to provide the accurate interface information when the binding is coupled with large conformational changes. In these cases, conformational change may induce chemical shift changes of the residues which are located far from interface. Nevertheless, chemical shift perturbation proved to be a simple and efficient method to get the interface information for transient complexes in which large conformational changes rarely happen. The
information obtained from chemical shift perturbation can be converted into ambiguous distance restraints and combined with docking methods to get a model of protein complex (48). The information of residues critical for the binding obtained from chemical shift mapping can be further validated through site-directed mutagenesis (49). The deuteration of the proteins combined with using TROSY-HSQC can extend the size limit of interaction system to above 100 kDa (50).
Cross saturation transfer
In some cases involved with large conformational changes, more accurate interface mapping can be obtained by cross saturation transfer (51). This method requires a deuteration of the protein with an interface to be identified, while its interaction partner is unlabeled. Thus, when combined with TROSY pulse, it can be used to accurately determine the interfaces of large complexes. The spins of aliphatic protons of the unlabeled protein (with chemical shift range) are first irradiated and saturated and then magnetization can be transferred through cross- relaxation to its deuterated interaction partner whose backbone amide signals are recorded by TROSY-HSQC pulse scheme. If the proton density of the doubly labeled molecule is sufficiently low, only residues in the interface show reduced intensities because the magnetization transfer is only limited to very nearby spins.(52).
Nuclear Overhauser Enhancement
When two nuclear spins are close enough (< 5 Å), the nuclear polarization can be transferred from one spin to the other through Nuclear Overhauser Enhancement (NOE), which provides the basis of protein structure determination by solution NMR. NOE remains to be the most accurate NMR method to determine a protein complex structure in liquid state. The intermolecular NOEs across an interface can be detected with differential
labeling strategy combined with isotope editing pulse schemes in order to discriminate from intra NOEs (53). Pair-wise intermolecular NOEs can be further converted into distance restraints based on the r-6 proportionality. A large number of intermolecular NOEs are needed to precisely determine the complex structure, however, which makes complex structure determination by intermolecular NOE method labor-intensive. In addition, for most weak, transient complexes, intermolecular NOE is generally difficult to observe because of the small and dynamic interfaces, although the solution structure of an ultra-weak complex was reported recently (54) .
Paramagnetic relaxation enhancement
A large number of metalloproteins contain paramagnetic metal ions which possess unpaired electrons. The unpaired electron of the metal center induces extra fluctuating magnetic fields at the nuclei of the protein. The external fluctuating magnetic fields contribute to relaxation of a nuclear spin of proteins by affecting its spin transitions. The paramagnetic relaxation enhancement (PRE) is proportional to r-6 (r is the distance between paramagnetic center and the nucleus), which is analogous to NOE. In some cases, these effects hinder the structure determination of high resolution structure of metalloproteins, because the existence of the metal ions causes fast relaxation of NMR signals of the surrounding nuclei. On the other hand, PRE can be converted into long-distance restraints to facilitate structure determination, especially for the observable nuclei which are relatively far from the metal center. The main advantage of PRE over NOE is that PRE can provide long-range distance (up to 35 Å) restraints (55). This is extremely useful for structure determination of complexes
(56).
PRE effects were used originally for the structural studies of metalloproteins that bear an intrinsic paramagnetic center. Recently, external spin label reagents were designed and site-specifically attached to proteins to extend the application
of PRE effects to diamagnetic proteins for structural and dynamic studies of transient complexes (57,58).
Free spin label or other PRE reagents can also be used to probe the exposed surface of proteins and hydration by detecting the PRE of the surface residues when the spin label is added to the protein solution (59,60). This method can also be extended to protein-protein interaction studies by detecting the protection effect of the complex formation against the PRE effect (61).
Pseudocontact shift
The time-averaged dipolar interaction between the unpaired electron and the nuclei has a contribution to the chemical shift if the interaction is anisotropic.
The observed chemical shifts (δ) for nuclei in metalloproteins contain three components
pcs con
dia
δ δ
δ
δ
= + +δdia is the diamagnetic chemical shift, δcon is the contact shift which is significant only for the nuclei with scalar coupling with the metal ion, and δpcs is the pseudocontact shift (PCS) resulting from the dipolar coupling between the
Nucleus M=metal
z
y
x
Ω θ
r
M
Figure 1.7. The coordinate system used to describe pseudocontact shift
unpaired electron of the metal ion and nuclei of protein. PCS is given by the equation 1.1 (figure 1.7)
( ) ( )
⎥⎦⎤⎢⎣⎡Δ − + Δ Ω
= sin cos2
2 1 3 cos 12 3
1 2 2
3 χ θ χ θ
πr ax rh
PCS (1.1)
Where r, θ and Ω are the polar coordinates of the nucleus in the reference frame of the magnetic susceptibility tensor (χ) which has axial (Δχax) and rhombic (Δχrhombic) components. If the structure of the protein as well as the orientation of the χ tensor frame and the sizes of the χ components are known, the δpcs can be predicted. Conversely, with a sufficient number of δpcs measured for nuclei in a protein with known structure, the orientation and size of the tensor can be calculated, using a five-parameter fit.
PCS can provide long-range distance restraints for the structure determination of single proteins or to determine the relative orientation of two components of a protein complex. The first example of solution structure determination of redox complex by intermolecular PCS is the structure of transient complex formed between cytochrome f and plastocyanin (62).
Restraints from the residual dipolar coupling
The first demonstration of the feasibility of measuring anisotropic dipolar coupling in solution was for cyanometmyoglobin, using the spontaneous alignment of this metalloprotein in a high magnetic field due to the large magnetic susceptibility anisotropy (63). Shortly after, weak alignment of other proteins in solution was achieved by different external alignment media with the introduction of anisotropic tumbling of macromolecules in solution. Residual dipolar coupling (RDC) can be described by the equation 1.2
( )
⎟⎠
⎜ ⎞
⎝
⎛ − + Ω
−
= sin cos2
2 1 3 cos
3 2 2
3
θ R θ
r D A
ij ax
res (1.2)
Where Aax and R are axial and rhombicity parameters of the alignment tensor, respectively, rij is the internuclear distance, θ and Ω determine the internuclear vector orientation with respect to the principle axes of the alignment tensor.
When the alignment is achieved through magnetic susceptibility anisotropy, the equation can be expressed as equation 1.3 (figure 1.8)
( )
⎟⎠
⎜ ⎞
⎝
⎛Δ − + Δ Ω
−
= sin cos2
2 1 3 cos 16 3
15
2 2
3 3 2
0
χ θ χ θ
π γ γ
rh ax
ij j res i
r h kT
D B
(1.3)Where B0 is the applied magnetic field, γi andγj are the gyromagnetic ratios of
nuclei i and j respectively, r is the i-j inter-nuclear distance, θ and Ω determine the inter-nuclear vector orientation with respect to the principle axes of the magnetic susceptibility tensor and Δχax and Δχrh are the axial and rhombic
z
y
x
Ω θ
r
iji
j
Metal
Figure 1.8. The coordinate system for the description of metal-aligned residual dipolar couplings.
components of the magnetic susceptibility tensor respectively, k is Boltzmann constant, h is Planck constant and T is absolute temperature.
Whereas NOE provide distances, RDCs provide angular information for the structure determination. Theoretically, de novo structure determination by complete set of RDCs is possible. But currently, RDC is mainly used for structure refinement because a set of residual dipolar couplings does not uniquely describe an inter-nuclear vector orientation. The other most obvious application of RDC is that it can accurately define the relative orientations of subunits in multiple-domain proteins or protein-protein complex. When combined with chemical shift perturbation data and other docking algorithms, it provides a fast but reliable means to determine the complex structure (64,48). In addition to its use in structure determination, RDC is also found to contain rich information on the dynamics of proteins or protein complexes. RDCs are averaged by motions spanning in a time scale from pico seconds to milli seconds, whereas 15N relaxation analysis is limited to motions from pico to nano second scales (65)(66).
Paramagnetic tagging
In recent years, the long-range effects of paramagnetism-based restraints from metallo-protein have drawn increasing attention for structural biologists. To apply those restraints to diamagnetic proteins, various paramagnetic tags were developed (67,68,69,70,71,72) . In order to maximize the utility of a paramagnetic probe, an ideal paramagnetic probe has to satisfy the following standards. The rigid attachment of a probe to protein can minimize the internal dynamics, thus induce large paramagnetic effects and avoiding averaging. Multiple attachment points are also required to provide more restraints for structure modeling.
The paramagnetic probes can be attached to a few sites of an unlabeled, large protein while the intermolecular paramagnetic effects can be detected on the labeled smaller interaction partners (73). Combined with TROSY-HSQC, ample
intermolecular restraints from paramagnetic tagging make it possible to determine structures of large complexes even at low concentrations, which offers a new avenue for structure determination of membrane protein complexes.
Thesis outline
The aim of this research is to study the dynamical and structural aspects of the transient complexes formed by iron sulfur proteins and their interaction partners, in order to advance the fundamental understanding of the mechanism of transient protein-protein interaction.
Chapter 2 describes the characterization of the dynamics of the complex formed between yeast Cc and bovine Adx using solution scattering and various paramagnetic NMR methods.
In the chapter 3, a new NMR method, RDC induced by an external paramagnetic probe is applied to the study of the intermolecular dynamics of the complex Cc/Adx.
Chapter 4 describes the complete interface mapping on Synechosystis Fd in the complex Fd/FTR by chemical shift perturbations and gallium substitution. The solution structure of Ga substituted Fd is further described in chapter 5.
Chapter 6 is about the study of ternary complex of Fd/FTR/Trx by NMR titration and docking using the paramagnetic restraints from the 4Fe-4S cluster of FTR.
Finally, conclusions are presented about the complexes of Cc/Adx and Fd/FTR/Trx. The prospects of paramagnetism-based restraints from natural metal center and external paramagnetic probes are discussed.
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