Stable folding core in the folding transition state of an alpha-helical integral membrane protein.

Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding.

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.

Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle.

An homology model of Candida methylica formate dehydrogenase (cm FDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (ps FDH) structure. In wild type cm FDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by -4 kcal mol(-1).

Cloning, sequence and expression of the lactate dehydrogenase gene from the human malaria parasite, Plasmodium vivax.

Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria. To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax ( PvLDH) Belem strain. The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E. coli. Structural differences between human LDHs and PfLDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs. The sequence similarity between PvLDH and PfLDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.