Protein and Peptide Letters - Volume 15, Issue 2, 2008

This issue of Protein and Peptide Letters (PPL) features two papers from the team of Alfredo Tomasselli and Bob Heinrikson and their colleagues [119-143]. Both of these contributions are slightly longer than typical PPL papers and both deal with the same enzyme, BACE, or β-secretase, a target for drug discovery for treatment of Alzheimer's Disease. What is the justification for taking up this much room in an issue of PPL? In the current era of rapidly paced discovery and publication, it is all too often the case that papers contain only the sketchiest of details of the experiments described. Many graduate students and their faculty mentors are frustrated at attempting to reproduce the experiments described in a paper, only to discover that essential elements of the experimental design have been left out, either due to deliberate obfuscation or to editorial demands for compression. This can lead to needless delays and considerable confusion for both student and professor. The two manuscripts presented here take the opposite tack and present the entire story of the expression, purification, characterization, and crystallization of BACE. One manuscript presents the expression in CHO cells, rapid purification using affinity methods, and strategies to reduce heterogeneity of the protein products to permit crystallization. The second manuscript presents expression in E. coli, followed by refolding and purification to yield crystallizable material. In each case, experimental protocols are described fully and analytical methods applied carefully to provide a complete picture of the status of the derived proteins. I firmly believe that these two contributions represent good examples for all laboratories in how to conduct and describe biochemical experiments. While I do not wish to encourage excessively long submissions to PPL, I believe that these papers can and should be used as examples for students in the research lab. I will make these available to all my students and ask that they follow the lead of these authors in writing reports on their work. I hope that others will find these useful as well.

BACE, or β-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid β peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)6 and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr1, and a derivative missing the first 24 amino acids beginning with E25. These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 Å resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F39-V40 bond, leaving the V40EM-ES432 (His)6 derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 Å resolution.

BACE (β-site APP cleaving enzyme) or β-secretase, the enzyme responsible for processing APP to give the Nterminal portion of the Aβ peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a Cterminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser432) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV....QTDES432), (2) pQE80L-Met-R-G-S-(His)6-G-S-I-E-T-D-(T1QH...QTDES432), and (3) pQE70-Met-BACE (R36GSFVEMG....PQTDES432(His)6) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 Å resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

With rapid increase in influenza A virus database, an important issue is whether the predictions are similar based on different datasets. Here we stratify 482 H3N2 hemagglutinins from influenza A virus in North America to different datasets. The predictions are made using logistic regression. The results show that the different datasets have significant impact on the predictions.

His-tagging is commonly used to aid and expedite the purification of recombinant proteins. It is commonly assumed, though less frequently tested, that the His-tag affects neither the structure nor the stability of the protein. Alanine:glyoxylate aminotransferase (AGT) is a peroxisomal pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the transamination of alanine and glyoxylate to pyruvate and glycine. AGT is a clinically relevant enzyme whose deficiency causes an inherited rare metabolic disorder named primary hyperoxaluria type I. Until now, the structure and function of this enzyme have been studied using recombinant wild-type AGT and variants purified using a hexahistidine tag. However, the study of the functional roles of the N- and C-termini in the dimerization process and on the import into the peroxisome, respectively, requires the preparation of human liver AGT without histidine tags. We report for the first time the expression of untagged AGT together with a new rapid protocol for its purification. In addition, the kinetic parameters for the forward and reverse transamination catalyzed by untagged AGT as well as the spectroscopic features, the KD(PLP), the pH and thermal stability of the enzyme in the holo- and apo-form have been determined. This investigation will be the starting point for a detailed understanding of the contributions of the N- and C-termini on the dimerization and folding of AGT, and on its import into the peroxisome. This is prerequisite to understand how pathological mutations affect the proper native quaternary and tertiary structure, stability, and targeting of the enzyme.

We have carried out a systematic analysis in order to evaluate whether Intra-Chain Disulfide Bridged Peptides (ICDBPs) observed in proteins of known three-dimensional structure adopt structurally similar conformations as they may correspond to structural/functional motifs. 406 representative ICDBPs comprising between 3 to 17 amino acid residues could be classified according to peptide sequence length and main-chain secondary structure conformation into 146 classes. ICDBPs comprising 6 amino acid residues are maximally represented in the Protein Data Bank. They also represent the maximum number of main-chain secondary structure conformational classes. Individual ICDBPs in each class represent different protein superfamilies and correspond to different amino acid sequences. We identified 145 ICDBP pairs that had ≤ 0.5 Å root mean square deviation value corresponding to their equivalent peptide backbone atoms. We believe these ICDBPs represent structural motifs and possible candidates in order to further explore their structure/function role in the corresponding proteins. The common conformational classes observed for ICDBPs defined according to the main-chain secondary structure conformations; H (helix), B (residue in a isolated beta bridge), C (coil), E (extended beta strand), G (310 helix), I (pi helix), S (bend), T (hydrogen-bonded turn) were; “CHHH”, “CTTC”, “CSSS” and “CSSC” (for ICDBP length 4), “CSSCC” (length 5), “EETTEE”, “CCSSCC”, “CCSSSC” (length 6), “EETTTEE” (length 7), “EETTTTEE” (length 8), “EEEETTEEEE” (length 10), “EEEETTTEEEE” (length 11) and “EEEETTTTEEEE” (length 12).

P5 (KWKKLLKKPLLKKLLKKL-NH2) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K3K4L5L6-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.

Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.

In a permeablized cell system, oxidized cyt c is able to induce caspase cascade whereas reduced cyt c cannot. In in vitro experiments, oxidized cyt c can promote H2O2 generation. It is suggested that the redox state of cyt c might regulate the initiation of apoptosis via regulation of cellular ROS level.

We have observed that hamster prion protein (PrPC) undergoes conformational changes on exposure to heat or sonication. If a sonication induced new conformer is seeded with a small amount of its abnormal pathogenic isoform (PrPSc) it undergoes a significant conversion to a proteinase-resistant isoform. This suggests the presence of a third stable PrP conformer, which may be intermediate in the conversion of PrPC to PrPSc.

Bacillus licheniformis ??-amylase (BLA) is routinely used as a model thermostable amylase in biochemical studies. Its starch hydrolysis activity has recently been studied in Tris buffer. Here, we address the question that whether the application of Tris buffer may influence the results of BLA activity analyses. Based on the inhibition studies and docking simulations, we suggest that Tris molecule is a competitive inhibitor of starch-hydrolyzing activity of BLA, and it has a high tendency to bind the enzyme active site. Hence, it is critically important to consider such effect when interpreting the results of activity studies of this enzyme in Tris buffer.

The current article describes the biophysical characterization and folding studies of fibroblast growth factor homologous factor-1b (FHF-1b) in comparison with acidic fibroblast growth factor (FGF-1). Our data indicates that FHF- 1 is significantly more stable than FGF-1. The folding mechanism of these two proteins seems to be different although they share high degree of sequence and structural similarity. FHF-1 unfolds through stable intermediate state while unfolding of FGF-1 is two-state. Interestingly, low concentration of sodium dodecyl sulfate (SDS) drives the folding pathway of FHF-1b to two-state.

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly- Cys amide, Ac-Tat48-60-Gly-Cys-NH2, having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat48-60-Gly-Cys-NH2 peptide exhibited excellent gene transfer efficacy in B16BL6 cells.

The number of atom-atom contacts in long distance can fit to the experimental binding energies in a dataset containing 151 experimental data with the correlation coefficient about 0.68. Based on this factor, a set of distancedependent empirical potentials for various types of short-distance (2.4 Å-5 Å) contacts was obtained by guided fitting, i.e. a set of two parameters fitting. Incorporation of these short-distance potentials improved the correlation coefficients to 0.881.

Oxyntomodulin is a proglucagon-derived gut hormone that reduces food intake and body weight, thus represents a potential therapy for obesity. We synthesized and crystallized oxyntomodulin. The crystal diffracts x-ray to 2.4 Å resolution and belongs to space group P213 with unit-cell parameters a=b=c= 48.44 Å, α=β=γ=90°. Preliminary analysis indicates a trimer packing in one asymmetric unit.

To elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism of the enzymes belonging to the haloacid dehalogenase (HAD) superfamily, TON_0559, a putative HAD subfamily protein from a hyperthermophilic archaeon Thermococcus onnurineus NA1, was expressed, purified and crystallized. X-ray diffraction data were collected to 2.0 Å resolution. The space group is C2, with unit cell parameters a = 121.2 Å, b = 62.9 Å, c = 37.5 Å and β= 106.5°.