Detergent-protein and detergent-lipid interactions : implications for two-dimensional crystallization of membrane proteins and development of tools for high throughput crystallography

Abstract

2.1 Scope of this Thesis This thesis represents an attempt to enlighten the role of the detergent in reconstitution and more specifically in two-dimensional (2D) crystallogenesis of membrane proteins. The construction of a tool for precise and routine measurements of detergent concentrations provided a valuable tool for better understanding and controlling the detergent issue. Additionally, a novel approach for detergent removal in 2D crystallization, i.e. the use of cyclodextrins was explored and a nanoliter dispensing high throughput tool was developed allowing for profound and sophisticated screening of optimal conditions for protein reconstitution and crystallization. 2.2 Combining Electron Microscopy and Atomic Force Microscopy Although electron crystallography has proven to be a powerful approach to structure determination of membrane proteins (for a recent example see (Gonen et al., 2005)) successes are somehow restricted to certain classes of membrane proteins (e.g., outer membrane porins, aquaporins, naturally occurring crystalline proteins). This is mainly due to the stability of these proteins with respect to biochemical manipulation. One can not exclude however, that these are simply more amenable to crystallization due to the nature of their molecular surfaces. 2D crystallization exhibits several advantages compared to 3D crystallization of membrane proteins: The simple fact that the proteins are allowed to reside in a native-like environment, i.e., the membrane and that their function is not impaired by the lateral crystal contacts is of considerable interest. If structural investigations shall not be restricted to static snapshots of different conformations and moreover structure-function relationships shall be established, then electron microscopy (EM) in combination with atomic force microscopy (AFM) surely represent a valuable approach. In Chapter 2 the combination of such data has been successfully applied to the ammonium transporter AmtB from Escherichia coli. The aim was to determine the crystal packing of the double-layered 2D crystals of AmtB by AFM in order to process the cryo EM data. Additionally, the AFM images, due to their outstanding signal-to-noise ratio, enabled the direct visualization of trimers in the reconstituted membranes. The topographical data from the AFM allowed the assessment of a single layer within the double layered crystals. 2.3 Investigating the Role of the Detergent In Chapter 3 the development of a fast and precise method for detergent concentration determination is presented. The robustness and wide application range of this method has been demonstrated by comparing concentrations of radioactively labeled dodecyl-[beta],D-maltoside (DDM) with measured contact angles, by measuring the amount of DDM bound to the proton/galactose symporter GalP from E. coli, by measuring the effects of 100 mM NaCl on the cmc of dodecyl-N,Ndimethylamine- N-oxide, by characterizing the surface energy of Parafilm, and finally by revealing the stoichiometry of complex formation between methyl-[beta]-cyclodextrin (MBCD) and different de- tergents. The possibility of performing such measurements routinely in membrane biochemistry is unique compared to all other methods available to date. Chapter 4 addresses the major aspects of detergent use in membrane protein purification and crystallization. First, the stability of GalP in different detergents is assessed, unveiling profound differences in the capacity of detergents to keep the protein in solution. Second, it is demonstrated, that the amount of a detergent, i.e., dodecyl-�,Dmaltoside, bound to a protein can be controlled during purification. At last the amount of different detergents for solubilization of E. coli lipids is determined, showing differences in the mechanisms by which detergents promote solubilization. Banerjee et al. (Banerjee et al., 1995) examined the preferential affinity of detergents for different lipids in mixed membranes (such as biological membranes). They showed that different detergents extract the serotonin 5-HT1A receptor from native membranes along with different lipids. The effect is considerable and might explain why different detergents exhibit such a different ability to keep a protein in its native state, because some might simply not be able to co-solubilize native lipids essential for the stability (and function) of the protein. The amount of detergent bound to a protein is of special interest when using dialysis or dilution for detergent removal. Furthermore, in most cases the protein must not be exposed to excess detergent which anyway fails to satisfactorily mimic the native bilayer. As pointed out in the discussion of Chapter 4, protein reconstitution is facilitated when the detergent collar that is present around the hydrophobic region of membrane proteins in solution is near its solubility limit (Psol). The same is true for the lipid: Reconstitution is likely to happen when liposomes are forming, therefore an excess of detergent is not desirable either. Additionally, even detergents known to have adverse effects on protein stability can be used for lipid solubilization, given that they are present at a minimal concentration. The use of detergent mixtures in crystallization can also have the effect of reducing the size of the detergent collar around the protein. Moreover, the free detergent concentration in detergent mixtures is altered by the presence of the second species and can be crucial to the formation of crystals in some cases (Koning, 2003). When using minimal amounts of detergent in a crystallization mixture, special care should be taken with respect to the formation of ternary micelles. Ideally, equilibration of the ternary mixtures prior to detergent removal needs to be completed. 2.4 The Use of Cyclodextrins for High Thorughput 2D Crystallization of Membrane Proteins Chapter 5 demonstrates the feasibility of the cyclodextrin-based detergent removal for twodimensional crystallization. The possibility of choosing different kinetics, simply by adding different amounts of cyclodextrin at various time intervals is one of the major advantages of this method. By implementing optical spectroscopy, it would be possible to slow down the detergent removal rate at the onset of proteoliposome and 2D crystal formation. As pointed out by Lichtenberg et al. (Lichtenberg et al., 2000) the rate of detergent removal has to be slow enough to allow for detergent-induced vesicle size growth, a process which is usually quite slow. This aspect is important to keep in mind as one defines the rate of detergent neutralization (in contrast to dialysis). At a first glance one might think that in this respect the cyclodextrin approach bears no advantage compared to dialysis. However, the rate of low-cmc detergent removal using dialysis can be too slow, thereby keeping the protein out from its native environment for too long, ultimately promoting its precipitation. In Chapter 6 we present an apparatus for parallel quantitative reconstitution and 2D crystallization of membrane proteins. Cyclodextrin provides a unique opportunity for high throughput implementation compared to other methods available today. Protein concentrating through controlled evaporation with concomitant detergent neutralization (to prevent detergent concentrating) is advantageous compared to commercially available protein concentrating devices which very often concentrate detergent micelles too. Moreover, the possibility of using one protein preparation for wide screening ensures that inconsistencies in results arising from preparative differences are excluded. Often, the detergent and lipid concentration of the purified protein are ill characterized, and this variability may be a cause for much of the irreproducibility and failure in crystallization (Wiener, 2004). So far the use of wide screening matrices (sparse matrix design) in 2D crystallography was restricted by the enormous number of experiments and amount of protein needed for a rigorous screening. The presented machine makes it possible to partially compensate for the first bottleneck in protein structure elucidation, which is the over-expression of membrane proteins. Fig. 2.1 summarizes the screening strategy based on the criteria discussed in Chapter 6 and above. Screening efficiency is provided by the subdivision of the problem into multiple subproblems and by their sequential screening. With the high throughput approach however, a new bottleneck arises as one will produce a large number of crystallization trials, which have to be screened for their outcome. Therefore –in analogy to the x-ray community– the development of automated sample preparation and automated electron microscopic analysis would provide substantial support to the 2D crystallographer. Combining step-by-step identification of key values necessary for crystallization (and/or efficient reconstitution) together with high throughput screening matrices opens up new prospects in the en deavor to membrane protein structure and function determination. Now it is possible to apply a semi-rational screening strategy and this might contribute to transform 2D crystallization from art to science (Jap et al., 1992)