Plasmodium falciparum triosephosphate isomerase: new insights into an old enzyme

Triosephosphate isomerase (TIM), a central enzyme in the glycolytic pathway, has been the subject of extensive structural and mechanistic investigations over the past 30 years. The TIM barrel is the prototype of the (β/α)8 barrel fold, which is one of the most extensively used structural motifs in enzymes. Mechanistic studies on TIM from a variety of sources have emphasized the importance of loop 6 dynamics for enzyme activity. Several conserved residues in TIM have been investigated by extensive site-directed mutagenesis of the enzyme from yeast, chicken, and trypanosoma. The cloning and sequencing of the TIM gene from the malarial parasite Plasmodium falciparum in 1993 revealed the unexpected mutation of a hitherto conserved residue serine (S96) to phenylalanine (F96). Subsequent results from the genome sequencing programs of Plasmodium falciparum, Plasmodium vivax, and Plasmodium yoelii confirmed the presence of the S96F mutation in malarial parasites. The crystal structure of PfTIM and several inhibitor complexes, including a high-resolution (1.1 Å) structure of the PfTIM 2-phosphoglycerate complex, revealed that loop 6 had a propensity to remain open, even in several ligand bound structures. Furthermore, both open and closed forms could be characterized for the same complex. Since glycolysis is the primary source of ATP for the malarial parasite during the intraerythrocytic stage, glycolytic enzymes present themselves as potential targets for inhibitors. Two distinct approaches have been explored. The use of dimer interface peptides, which interfere with assembly, has proved promising. Inactivation of the enzyme by modification of a cysteine (C13) residue, which lies close to the active site residue, lysine (K12) is another potential strategy. The differential reactivity, of the four-cysteine residues, at positions 13, 126, 196, and 217 in each subunit has been established using electrospray ionization mass spectrometry. Studies of single tryptophan mutants (W11F and W168F) of PfTIM provide a probe to study folding, stability, and inhibitor interactions

Inhibition of Plasmodium falciparum triose-phosphate isomerase by chemical modification of an interface cysteine- electrospray ionization mass spectrometric analysis of differential cysteine reactivities

Plasmodium falciparumtriose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 is > Cys-13 » Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed

Conformational and Sequence Signatures in β Helix Proteins

Summaryβ helix proteins are characterized by a repetitive fold, in which the repeating unit is a β-helical coil formed by three strand segments linked by three loop segments. Using a data set of left- and right-handed β helix proteins, we have examined conformational features at equivalent positions in successive coils. This has provided insights into the conformational rules that the proteins employ to fold into β helices. Left-handed β helices attain their equilateral prism fold by incorporating “corners” with the conformational sequence PII-PII-αL-PII, which imposes sequence restrictions, resulting in the first and third PII residues often being G and a small, uncharged residue (V, A, S, T, C), respectively. Right-handed β helices feature mid-sized loops (4, 5, or 6 residues) of conserved conformation, but not of conserved sequence; they also display an α-helical residue at the C-terminal end of L2 loops. Backbone conformational parameters (ϕ,ψ) that permit the formation of continuous, loopless β helices (Perutz nanotubes) have also been investigated

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single-tryptophan mutants

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-Å crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid

Circular dichroism of designed peptide helices and β-hairpins: analysis of trp- and tyr-rich peptides

VCD versus ECD spectroscopy. Peptides rich in aromatic residues yield anomalous far-UV electronic circular dichroism (ECD) spectra that preclude secondary structure assignment. The utility of vibrational circular dichroism (VCD) in conformation analysis is demonstrated by using a set of well-defined peptide helices and hairpins containing proximal aromatic residues

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CαH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides

Molecular conformation and packing of peptide hairpins in the solid state: Structures of two synthetic octapeptides containing 1-aminocycloalkane-1-carboxylic acid residues at the i+2 position of the βturn

Peptide β-hairpin formation is facilitated by centrally positioned D-Pro-Xxx segments. The synthetic peptides Boc-Leu-Phe-Val-D-Pro-Ac6c-Leu-Phe-Val-OMe (1) and Boc-Leu-Phe-Val-D-Pro-Ac8c-Leu-Phe-Val-OMe (2) were synthesized in order to explore the role of bulky 1-aminocycloalkane-1-carboxylic acid residues (Acnc, where n is the number of carbon atoms in the ring), at the i+2 position of the nucleating β turn in peptide β hairpins. Peptides 1 and 2 crystallize in the monoclinic space group P21 with two molecules in the asymmetric unit. The crystal structures of 1 and 2 provide conformational parameters for four peptide hairpin molecules. In all cases, the central segments adopts a type II′ β-turn conformation, and three of the four possible cross-strand hydrogen bonds are observed. Fraying of the hairpins at the termini is accompanied by the observation of NH...∏ interaction between the Leu(1)NH group and Phe(7) aromatic group. Cross strand stabilizing interactions between the facing residues Phe(2) and Phe(7) are suggested by the observed orientation of aromatic rings. Anomalous far-UV CD spectra observed in solution suggest that close proximity of the Phe rings is maintained even in isolated molecules. In both peptides 1 and 2, the asymmetric unit consists of approximately orthogonal hairpins, precluding the formation of a planar β-sheet arrangement in the solid state. Solvent molecules, one dioxane and one water in 1, three water molecules in 2, mediate peptide association. A comparison of molecular conformation and packing motifs in available β-hairpin structures permits delineation of common features. The crystal structures of β-hairpin peptides provide a means of visualizing different modes of β-sheet packing, which may be relevant in developing models for aggregates of polypeptides implicated in disease situations

Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy

Objective: Protein glutathionylation is considered an important post-translational modification in the pathogenesis of complex diseases. The aim of this study was to examine whether hemoglobin (Hb) is modified by reduced glutathione (GSH) via oxidation of the thiol groups present in diabetes and its associated microangiopathy and to determine whether oxidative imbalance has any correlation with glutathionylated Hb (HbSSG) levels. Methods: The study group consisted of a total of 130 subjects which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. All subjects were assessed for glycemic and lipidemic status, while diabetic subjects were also assessed for the diagnosis of retinopathy and nephropathy. RBC lysates from all the subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for HbSSG β-globin chains. Levels of GSH and thiobarbituric acid substances (TBARS) levels were measured by spectrophotometric and fluorimetric methods, respectively. Results: The positivity for HbSSG in diabetic subjects with microangiopathy was significantly higher (69%) compared to diabetics without microangiopathy (22%) and control subjects (14%). In univariate regression analysis, HbSSG levels were significantly associated with the duration of diabetes, HbA1c, and TBARS levels. GSH levels were negatively correlated (r = -0.57, P < 0.001) with HbSSG in diabetic subjects. A significant inverse correlation (r = -0.42, P < 0.001) between the GSH levels and HbA1c levels was also seen in diabetic subjects. Conclusions: This is perhaps the largest LC-MS-based study to demonstrate that HbSSG levels are markedly increased in diabetic subjects with microangiopathy. Since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased HbSSG concentrations and altered reduction-oxidation (redox) signaling

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5°C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 Å crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures

Designed peptides with homochiral and heterochiral diproline templates as conformational constraints

Diproline segments have been advanced as templates for nucleation of folded structure in designed peptides. The conformational space available to homochiral and heterochiral diproline segments has been probed by crystallographic and NMR studies on model peptides containing L-Pro-L-Pro and D-Pro-L-Pro units. Four distinct classes of model peptides have been investigated: a) isolated D-Pro-L-Pro segments which form type II β-turn; b) D-Pro-L-Pro-L-Xxx sequences which form type II′-I (βII′-I′, consecutive β-turns) turns; c) D-Pro-L-Pro-D-Xxx sequences; d) L-Pro-L-Pro-L-Xxx sequences. A total of 17 peptide crystal structures containing diproline segments are reported. Peptides of the type Piv-D-Pro-L-Pro-L-Xxx-NHMe are conformationally homogeneous, adopting consecutive β-turn conformations. Peptides in the series Piv-D-Pro-L-Pro-D-Xxx-NHMe and Piv-L-Pro-L-Pro-L-Xxx-NHMe, display a heterogeneity of structures in crystals. A type VIa β-turn conformation is characterized in Piv-L-Pro-L-Pro-L-Phe-OMe (18), while an example of a 5→1 hydrogen bonded α-turn is observed in crystals of Piv-D-Pro-L-Pro-D-Ala-NHMe (11). An analysis of pyrrolidine conformations suggests a preferred proline puckering geometry is favored only in the case of heterochiral diproline segments. Solution NMR studies, reveal a strong conformational influence of the C-terminal Xxx residues on the structures of diproline segments. In L-Pro-L-Pro-L-Xxx sequences, the Xxx residues strongly determine the population of Pro-Pro cis conformers, with an overwhelming population of the trans form in L-Xxx = L-Ala (19)