Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein.

α-Helical membrane proteins have eluded investigation of their thermodynamic stability in lipid bilayers. Reversible denaturation curves have enabled some headway in determining unfolding free energies. However, these parameters have been limited to detergent micelles or lipid bicelles, which do not possess the same mechanical properties as lipid bilayers that comprise the basis of natural membranes. We establish reversible unfolding of the membrane transporter LeuT in lipid bilayers, enabling the comparison of apparent unfolding free energies in different lipid compositions. LeuT is a bacterial ortholog of neurotransmitter transporters and contains a knot within its 12-transmembrane helical structure. Urea is used as a denaturant for LeuT in proteoliposomes, resulting in the loss of up to 30% helical structure depending upon the lipid bilayer composition. Urea unfolding of LeuT in liposomes is reversible, with refolding in the bilayer recovering the original helical structure and transport activity. A linear dependence of the unfolding free energy on urea concentration enables the free energy to be extrapolated to zero denaturant. Increasing lipid headgroup charge or chain lateral pressure increases the thermodynamic stability of LeuT. The mechanical and charge properties of the bilayer also affect the ability of urea to denature the protein. Thus, we not only gain insight to the long-sought-after thermodynamic stability of an α-helical protein in a lipid bilayer but also provide a basis for studies of the folding of knotted proteins in a membrane environment.

Developmental kinetics of bovine nuclear transfer and parthenogenetic embryos.

Early developmental kinetics of nuclear transfer (NT) embryos reconstituted with blastomeres and parthenogenones produced by ionophore activation followed by either dimethylaminopurine (DMAP) or cycloheximide (CHX) treatment was studied. In vitro produced (IVP) embryos served as controls. Embryos were cultured to the hatched blastocyst stage, and images were recorded every 0.5 h throughout the culture period. The longest cell cycle shifted from 4th to 5th cycle (26 +/- 4 and 44 +/- 5 h) in NT-embryos compared to IVP-embryos (41 +/- 2 and 20 +/- 3 h) and showed greater asynchrony between blastomeres than any other embryo category. Compared to DMAP, CHX prolonged the 1(st) (23 +/- 1 vs. 33 +/- 1 h) and shortened the 3(rd) cell cycle (17 +/- 2 vs. 13 +/- 1 h). Moreover, though cytoskeleton activity was initialised, a larger proportion of CHX embryos was unable to accomplish first cleavage. The parthegenones differed from IVP embryos with respect to the lengths of the 1st, 3rd, and 4th cell cycles and time of hatching. The findings are discussed in relation to known ultrastructural, chromosomal and genomic aberrations found in NT embryos and parthenogenones. We hypothesize that the shift of the longest cell cycle in NT embryos is associated with a shift in the time of major genomic transition.

Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media.

The kinetics of the in vitro development of early embryos from bovine zygotes derived in vitro and in vitro were compared, investigating the effect of serum during in vitro maturation and fertilization (IVM-IVF) and in culture. Zygotes were collected from superovulated heifers or produced in vitro from immature oocytes with or without serum supplementation, and cultured subsequently in defined culture medium (SOFaaci) with or without serum supplementation. Time-lapse images were recorded every 0.5 h throughout the culture period. More in vivo- than in vitro-derived zygotes developed to the compact morula or blastocyst stages (87% versus 47-54%, respectively; P < 0.05). Embryo development was blocked predominantly at the second or fourth cell cycles (28 and 29%). However, blastomeres degenerated at all cleavage stages. Serum supplementation during IVM-IVF resulted in abnormally sized blastomeres at first cleavage (defined serum: 20-22% versus in vivo-derived: 8%, P < 0.05). The duration of the second, third and fifth cell cycles of in vivo-derived zygotes were 1-5 h shorter compared with those of in vitro-derived zygotes cultured under similar conditions (P < 0.05). However, the kinetics of embryo development was affected by serum during IVM-IVF and culture. The first and fourth cell cycles were prolonged by 4-5 h in the absence of serum during IVM-IVF, whereas the presence of serum during culture decreased the duration of the fourth cell cycle and triggered premature blastulation. The results of this study illustrate the differences and similarities between the morphology and developmental kinetics of in vivo- and in vitro-derived zygotes, and show how serum supplementation during IVM-IVF and culture can alter these parameters.

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos.

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.

Manipulating the folding of membrane proteins: using the bilayer to our advantage.

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.

Can we identify the forces that drive the folding of integral membrane proteins?

Protein folding has been at the forefront of molecular cell biology research for several years. However, integral membrane proteins have eluded detailed molecular level study until recently. One reason is the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is nevertheless a large body of information on lipid properties and in particular on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to design efficient in vitro, lipid-bilayer folding systems for membrane proteins. Bacteriorhodopsin has been used as a model system for our initial studies: we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency seem to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer and the lateral pressure that the lipid chains exert on their neighbouring folding protein. These are generic properties of the bilayer that can be achieved with simple mixtures of many types of biological lipid and seem to be important in vivo.

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin.

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics.

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.

Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin.

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb.

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.

Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.

Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development of the system was based on results using parthenogenetic and in vitro fertilized zygotes in order to (a) select the optimal primary activation agent that induced the lowest lysis rate but highest parthenogenetic blastocyst yield, (b) evaluate the quantity and quality of zona-free blastocysts produced in WOWs, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20.8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1%) blastocysts per successfully fused and surviving reconstructed embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1%) recipients pregnant at 5 months after transfer. These results suggest that the zona-free nuclear transfer technique generates blastocysts of equivalent quantity and quality compared to conventional micromanipulation methods, requires less technical expertise, is less time consuming and can double the daily output of reconstructed embryos (even after taking into consideration the rejection of the half oocytes containing the metaphase plate).

Application of the zona-free manipulation technique to porcine somatic nuclear transfer.

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.

Folding, assembly, and stability of the major light-harvesting complex of higher plants, LHCII, in the presence of native lipids.

The influence of thylakoid lipids on the association kinetics and thermal stability of the major light-harvesting complex of photosytem II (LHCII) has been studied in vitro. The apoprotein, light-harvesting chlorophyll a/b-binding protein (Lhcb1), can be refolded and complexed with pigments in detergent solution even in the absence of lipids. Two thylakoid lipids, phosphatidyl glycerol and digalactosyl diacylglycerol, are known to interact specifically with LHCII in vivo. Here we show that both of these lipids, as well as monogalactosyl diacylglycerol, stabilize reconstituted LHCII toward thermal denaturation. Two slow kinetic phases are connected with the establishment of energy transfer between chlorophyll b and chlorophyll a and, thus, are thought to reflect the formation of the pigment-protein complex with tightly coupled chlorophylls. The lipids studied here all have the same effect on the rate of complex assembly in vitro and slow these two kinetic phases by the same degree. Both kinetic phases also slow when reactant concentrations are decreased, suggesting that the corresponding reaction step(s) involve(s) pigment binding.

Estimations of lipid bilayer geometry in fluid lamellar phases.

The excess water bilayer thickness, d(l,0), and molecular area, A(0), of lipid amphiphiles in the fluid lamellar phases of dioleoylphosphatidylcholine (DOPC) and dipalmitoleoylphosphatidylcholine (DPolPC) have been estimated between 15 and 50 degrees C and for dimyristoylphosphatidylcholine (DMPC) between 25 and 50 degrees C. These determinations have been made from X-ray measurements on samples of known water composition. With respect to temperature, T, d(l,0) and A(0) are well fitted to a linear equation. We find d(l,0) (A)=(35.68+/-0.02)-(0.0333+/-0.0006)T (degrees C) and A(0) (A(2))=(70.97+/-0.05)+(0.136+/-0.001)T (degrees C) for DOPC, d(l,0) (A)=(35.2+/-0.1)-(0.068+/-0.003)T (degrees C) and A(0) (A(2))=(59.7+/-0.2)+(0.210+/-0.006)T (degrees C) for DMPC, and d(l,0) (A)=(34.54+/-0.03)-(0.0531+/-0.0009)T (degrees C) and A(0) (A(2))=(67.12+/-0.09)+(0.173+/-0.003)T (degrees C) for DPolPC. The accuracy of these estimates depends largely on how accurately the excess water point is determined. Ideally, reliable X-ray and compositional data will be available around the excess water and it may be found by simple inspection, but this is the exception rather than the rule, since samples close to water excess normally sequester sizeable amounts of water in defects, which lead to an underestimate of d(l,0). and overestimate of A(0). In this paper, we report a methodology for identifying and removing such data points and fitting the remaining data in order to determine the excess water point.

Unravelling the folding of bacteriorhodopsin.

The folding mechanism of integral membrane proteins has eluded detailed study, largely as a result of the inherent difficulties in folding these proteins in vitro. The seven-transmembrane helical protein bacteriorhodopsin has, however, allowed major advances to be made, not just on the folding of this particular protein, but also on the factors governing folding of transmembrane alpha-helical proteins in general. This review focusses on kinetic and equilibrium studies of bacteriorhodopsin folding in vitro. It covers what is currently known about secondary and tertiary structure formation as well as the events accompanying retinal binding, for protein in detergent and lipid systems, including native membrane samples.

The final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH.

The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s-1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s-1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s-1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors.

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.