Subcellular location of the coupling protein TrwB and the role of its transmembrane domain.

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.

The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions.

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid. Previous studies indicated that the transmembrane domain of TrwB plays a role beyond the mere anchoring of the protein to the membrane. TrwB has also been shown to interact with other conjugative proteins, such as the VirB10-like protein of R388 TrwE. The goal of this study is to elucidate the role of the different domains of TrwB and TrwE in their biological function, and in both self- and TrwB-TrwE interactions. To this aim, a series of TrwB and TrwE deletion mutant proteins were constructed. Conjugation and interaction studies revealed that the transmembrane domain of TrwB, and particularly its second transmembrane helix, is needed for TrwB self-interaction and for R388 conjugative transfer and that there are contacts between TrwB and TrwE in the membrane. On the contrary, the lack of the TMD of TrwE does not completely abolish R388 conjugation although the interaction between TrwE-TrwB is lost. These results identify protein-protein interactions inside the membrane needed for T4SS function.

Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein.

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim. Biophys. Acta 1798(11), 2160-2169 (2010)] that TrwB can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for ATP. In the present contribution we examine the structural effects of membrane insertion on TrwB, by comparing the protein in reconstituted form and in the form of protein/lipid/detergent mixed micelles. TrwB was reconstituted in PE:PG:CL (76.3:19.6:4.1mol ratio) with a final 99:1 lipid:protein mol ratio. This lipid mixture is intended to mimic the bacterial inner membrane composition, and allows a more efficient reconstitution than other lipid mixtures tested. The studies have been carried out mainly using infrared spectroscopy, because this technique provides simultaneously information on both the lipid and protein membrane components. Membrane reconstitution of TrwB is accompanied by a decrease in ß-sheet contents and an increase in ß-strand structures, probably related to protein-protein contacts in the bilayer. The predominant α-helical component remains unchanged. The bilayer-embedded protein becomes thermally more stable, and also more resistant to trypsin digestion. The properties of the bilayer lipids are also modified in the presence of TrwB, the phospholipid acyl chains are slightly ordered, and the phosphate groups at the interface become more accessible to water. In addition, we observe that the protein thermal denaturation affects the lipid thermal transition profile.

Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein.

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The protein specificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity.

The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB.

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBDeltaN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBDeltaN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.

Purification of different lipases from Aspergillus niger by using a highly selective adsorption on hydrophobic supports.

In this manuscript, we have purified three different lipases from crude preparations from Aspergillus niger in a simple fashion, secluding the esterases and other enzymes presented in the preparation. Firstly, the crude was offered at low ionic strength to octyl agarose. The support specifically adsorbed two lipases, with molecular weights of 43 and 65 kDa. Desorption with a gradient of Triton X-100 permitted to fully purify both lipases. The addition of octadecyl-Sepabeads support to the non-adsorbed proteins on octyl-agarose permitted to selectively adsorb a third lipase, having a molecular weight of 31 kDa. Desorption of the enzyme using Triton X-100 permitted to have also a pure sample of this enzyme. A significant percentage of esterase activity remains in the supernatant, derived from esterases or lipases unable to become adsorbed on the employed supports. Furthermore, these purified lipases were immobilized via ionic adsorption on DEAE-Sepharose and their selectivity was analyzed in the kinetic resolution of (+/-)-O-2-butyryl-2-phenylacetic acid and (+/-)-mandelic acid methyl ester. In the resolution of (+/-)-O-2-butyryl-2-phenylacetic acid, the crude extract preparation gave a low enantioselectivity value (E = 9), whereas the three immobilized preparations of purified lipases exhibited an increase in E-value from 11 (43 kDa lipase) to > 100 (31 kDa lipase). When (+/-)-mandelic acid methyl ester was used, the crude extract preparation presented low enantioselectivity hydrolyzing the S enantiomer quicker, while the purified lipase preparations preferred the R one. In this case, the 65 kDa lipase was the most selective enzyme (E = 20).

Determination of protein-protein interactions through aldehyde-dextran intermolecular cross-linking.

A very simple strategy, based on the intermolecular cross-linking of associated proteins by using aldehyde-dextrans, has been proposed to detect protein-protein interactions. Aldehyde-dextran was able to cross-link different enzymes composed of several polypeptide chains (e.g., trypsin and penicillin G acylase), proteolyzated proteins (e.g., extracts from porcine pancreas) and finally, an immunocomplex (horseradish peroxidase/anti-horseradish peroxidase). This cross-linked immunocomplex could be selectively adsorbed on immobilized anti-rabbit IgG. The presence of unspecific covalent attachment between unrelated protein molecules was not detected. Thus, this strategy permits the cross-linking of different protein components and avoids the formation of nonspecific protein-protein associations.

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran.

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts.

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester [(+/-)-1] to produce the corresponding acid [2], the octyl-25KDa preparation showed the best enantioselectivity (E) value (E = 7) at pH 5 and 25 degrees C, whereas the phenyl-PPL was the most enantioselective (E = 10) at pH 5, 4 degrees C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (+/-)-2-O-butyryl-2-phenylacetic acid [(+/-)-3] with a high E value (E > 100); for example, with octadecyl-33 KDa enzyme at pH 8.