The 2.05-Å resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasite’s life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel.
Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cellfree expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.Keywords: cell-free protein expression; integral membrane proteins; structural genomics; high-throughput protein expression Integral membrane proteins (MPs), despite their biological importance, currently account for <1% of all known high resolution protein structures. MPs are notoriously difficult to work with, and expression, detergent solubilization, purification, and crystallization all present unique challenges over their soluble counterparts (White 2004). MPs generally express at much lower levels than soluble proteins and, when in vivo overexpression is successful, the protein can be cytotoxic or incorporated into insoluble inclusion bodies. Following successful MP expression, a suitable detergent condition must also be found that simultaneously extracts the protein from the membrane while retaining the native fold and function. This protein-detergent complex (PDC) is often heterogeneous, creating numerous problems in purification and crystallization. Optimizing purification, assaying protein function, and crystallization all require milligram quantities of protein, and MP expression is therefore a limiting step in macromolecular structure determination (Dobrovetsky et al. 2005;Eshaghi et al. 2005;Korepanova et al. 2005;Columbus et al. 2006;Surade et al. 2006). One recognized alternative is cell-free (CF) expression (Klammt et al. 2004). 4 These authors contributed equally to this work.
Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.
The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.
Ab initio and density functional calculations have been performed to gain a better understanding of the epoxide ring-opening reaction catalyzed by epoxide hydrolase. The S(N)2 reaction of acetate with 1S,2S-trans-2-methylstyrene oxide to provide the corresponding diol acetate ester was studied with and without general-acid catalysis. MP2 and DFT (B3LYP) calculations predict, for the noncatalyzed reaction, a central barrier of approximately 20-21 kcal/mol separating the reactants from products depending on which carbon center in the epoxide is undergoing attack. From these gas-phase reactions the immediate alkoxide products are not energetically far below their associated transition states such that the reaction is predicted to be endothermic. Inclusion of aqueous solvation effects via a polarizable continuum model predicts the activation barrier to increase by almost 10 kcal/mol due to the solvation of the acetate ion nucleophile. The activation barrier for the epoxide ring-opening reaction is reduced to approximately 10 kcal/mol when phenol, as the general-acid catalyst, is included in the gas-phase calculations. This is due to the immediate product being the neutral ester rather than the corresponding alkoxide. The transition state in the general-acid-catalyzed reaction is earlier than that for the noncatalyzed reaction and the reaction is highly exothermic. Molecular mechanics calculations of 1S,2S-trans-2-methylstyrene oxide in the active site of murine epoxide hydrolase show two possible binding conformations. Both conformers have the epoxide oxygen forming hydrogen bonds with the acidic hydrogens of the catalytic tyrosines (Tyr381 and Tyr465). These two conformations likely lead to different products since the nucleophile (Asp333-CO(2)(-)) is positioned to react with either carbon center in the epoxide.
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