Protein and Peptide Letters - Volume 10, Issue 4, 2003

Calmodulin (CaM) is an EF-hand Ca(II)-binding protein involved in the regulation of many important biological processes. To date, there is a wealth of information available concerning studies to obtain site-specific calcium binding affinities of CaM, and further to estimate the cooperativity of calcium binding using mutational studies, peptide models, and proteolytic fragmentation. In this paper, we will discuss the energetics of calcium binding and the strong relationship between calcium binding cooperativity and conformational change. We then explain the difficulty of studying key determinants of calcium binding affinity of CaM due to the large change of calcium binding affinity upon mutation. Subsequently, we will introduce “grafting” as a novel approach to obtain the site-specific metal binding properties of calmodulin.

The polyphemusins present in the hemocytes of the horsechoe crab and their structurally modified analogs have been shown to exhibit activity against HIV-1. Among the many variants, T22 ([Tyr5,12, Lys7]-polyphemusin II), and its shorter and more potent analog, T140 [Arg1-Arg-2-Nal-Cys-Tyr5- Arg-Lys-D-Lys-Pro-Tyr10-Arg-Cit-Cys-Arg14] (Polyphemusin II-derived peptide), affect the HIV-cell fusion process and inhibit the T-cell line-tropic (T-tropic) HIV-1 infection. Conformational studies of polyphemusin II derived peptide have been carried out by 1H and 13C 2D-NMR and MD simulations in water and HFA (40%). The NMR parameters of chemical shift, temperature coefficients of the NH chemical shifts, 3JNHa coupling constants and the pattern of nOe's were used to deduce the structural characteristics. Solution structures were generated using dihedral and distance restraints by MD simulations. The structures are characterized by a dominant family possessing an anti-parallel β-pleated sheet that is constrained by the disulphide bridge between Cys4 and Cys13. The two strands of the β-sheet are joined by a Type II' β-turn spanning the residues Lys7-D-Lys8-Pro9-Tyr10. This conformation is present in both water and HFA. The only difference in the two structures is that the β-strands are more cohesive in HFA being firmly held by H-bonds. The solution structures generated from MD simulations were refined by MARDIGRAS to R-factors of 0.44 and 0.57 in water and HFA respectively. The conformation deduced for T140 is very similar to that reported for T22 and is thought to be associated with their anti HIV activity.

Cry4B toxin is a mosquito-larvicidal protein from the Bacillus thuringiensis subsp. israelensis. We have investigated the role of two conserved hydrophobic residues of Cry4B in structural stabilization. Substitutions of the leucine-175 and isoleucine-189 on helix α5 with valine and leucine did not affect the expression level, solubility and proteolytic processing. Steady state analysis of an unfolding experiment as monitored by circular dichroism and fluorescence spectroscopy demonstrated a typical two-state transition. The determined unfolding free energy for the L175V mutant revealed a structural destabilization of 10.49 kcal / mol relative to the wild type. However unfolding kinetic analysis gave identical activation energy for wild type and both mutants. Our findings suggested that a perturbation on the close packing of the hydrophobic side chains in protein interior could lead to a significant destabilization of the native conformation.

An alternative method for expressing the recombinant proteins in Escherichia coli is proposed. Unlike the ordinary induction protocol where the cells in the early- to mid-log phase are induced for the protein production, this alternative protocol utilizes the cells in the stationary phase. By using a glutathione S-transferase fusion protein as an example, the protocol proposed in this report yielded a higher amount of the desired protein than that from the ordinary protocol. This protocol also suppressed the proteolytic cleavage of the desired protein in the Escherichia coli cytoplasm, thus it should be particularly useful to produce proteins that undergo unwanted cleavages.

The Cys69 residue of an Anopheles dirus glutathione S-transferase isoform (adGSTD3-3), was characterized to elucidate its contribution in both catalysis and structural support. Nine mutants were generated at this position by replacing the residue with polar, non-polar and charged residues. The polar residues changed the Vm of the enzymes. With non-polar residues, the enzymes were unable to fold and were expressed in the insoluble inclusion form. With charged residues, the soluble enzyme yields were only 3% of the wild type protein. Molecular dynamics simulation also was performed to understand the changes in the enzyme structure. These findings are additional evidence of the importance of structural residues that affect the enzymatic properties such as Vm, Km and enzyme specificity.

α-Hemoglobin fragments α-(133-141), α-(134-141), α-(135-141), α-(137-141), α-(134-140), α- (133-138), α-(134-140) and α-(137-138) stimulate L929 tumor cell proliferation, α-(134-141) being the most active. α-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells. In L929 cells the effect of α-(134-141) is cell density independent; in M3 cells α-(137-141) and α-(134-141) are most active at density 10,000 cells / well (96 well plate) independently on FBS content.

Transglutaminase (TGase) has been reported to be involved in the regulation of cell growth. We examined the effects of polyamines on TGase activity. The polymerization of casein was inhibited by putrescine (PUT) and spermidine (SPD). On the other hand, polymerization of N,N-dimethylcasein was increased by spermine (SPM) and SPD. These results suggested polyamines played two distinct roles as inhibitor and promoter for TGase-catalyzed protein polymerization.

The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.

The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4 1 2 1 2 or P4 3 2 1 2 and diffract to better than 2.2Å resolution.

The ubiquitous NAD+ synthetase catalyzes the key step in the biosynthesis of nicotinamide adenine dinucleotide. NH3-dependent NAD+ synthetase from Helicobacter pylori was purified to homogeneity and crystallized using PEG 1500 as a preciptant. The crystal diffracted up to a resolution of 2.3+ and was found to belong to space group C2 with unit cell dimensions of a = 93.8, b = 48.3, c = 64.2 Å and α = γ = 90, β = 110.0°.

Hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, was crystallized by the hanging drop vapor diffusion technique with a precipitant solution containing 10% polyethylene glycol 3350 and 50 mM HEPES-Na, pH 7.5. The Hb D crystals of G. gigantea, which diffract to at least a 2.0 Å resolution, belong to the monoclinic space group C2 with unit cell dimensions of a = 112.1 Å, b = 62.4 Å, c = 54.0 Å, and β = 110.3 . One αβ dimer molecule of Hb D existed in an asymmetric unit, with a calculated value of Vm of 2.77 Å3Da-1.