Novel semisynthetic method for generating full length beta-amyloid peptides.

Bacterial expression of full length beta-amyloid (Abeta) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semisynthetic method in which Abeta1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Abeta1-29-Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Abeta1-29-MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Abeta1-29-MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH2-A30C-Abeta30-40, which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Abeta1-29-MESNA. Native chemical ligation proceeds rapidly and efficiently (>90% yield) to give A30C-Abeta1-40. The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (>90% yield) to give native sequence Abeta1-40. Overall, this system is highly efficient, and can yield approximately 8-10 mg of pure Abeta1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Abeta peptides. We have also expressed an Abeta construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Abeta1-29-Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15N or 13C labels for NMR. Future work will also include extending this system to longer Abeta peptides, such as Abeta1-42.

Cold denaturation of monoclonal antibodies.

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. Tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from -5 to 50 degrees C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, DeltaG(u), at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. The Gibbs-Helmholtz expression fit to the DeltaG(u) versus temperature data from fluorescence gave a DeltaC(p) of 8.0 kcal mol(-1) K(-1), a maximum apparent stability of 23.7 kcal mol(-1) at 18 degrees C, and an apparent cold denaturation temperature (T(CD)) of -23 degrees C. DeltaG(u) values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T(CD). Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. Thus, mAbs have a potential to undergo cold denaturation at storage temperatures near -20 degrees C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.

Encapsulation and NMR on an aggregating peptide before fibrillogenesis.

The early stages of peptide and protein aggregation include the formation of soluble oligomers, some of which may be cytotoxic. There is a paucity of structural information on these oligomers, however, because they are temporally unstable and tend to aggregate further into insoluble protofibrils and fibrils. To obtain structural information on soluble oligomers, we have developed a procedure for encapsulating a fibril-forming peptide, Peptide 1 (NH2-SDDYYYGFGSNKFGRPRDD-COOH), in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine single bilayer vesicles (POPC SBVs). We also encapsulated a non-fibril forming peptide, Peptide 2 (NH2-EEWEE-COOH), in POPC SBVs. The nominal concentration of Peptide 1 in the resulting 40 nm diameter SBVs was 2.4 +/- 0.1 mM, well above the concentration at which Peptide 1 forms fibrils. We demonstrated that these peptides had indeed been encapsulated by measuring longitudinal relaxation times (T1) in the presence and absence of a paramagnetic substance, 1 mM Gd-EDTA, by NMR spectroscopy. When the peptides were free in solution, they showed the expected shortening of T1 times and broadening of NMR peaks. In contrast, peptide encapsulated in POPC SBVs were shielded from the effects of Gd-EDTA and showed preservation of T1 values and NMR line widths. To demonstrate that encapsulation inhibits fibril formation, we measured one-dimensional proton (1D-1H) NMR spectra of the peptides in solution, and of the encapsulated peptides immediately after encapsulation, and 4 days after encapsulation, because Peptide 1 forms fibrils within 1 day. A 2.8 mM solution of Peptide 1 shows the loss of NMR signal expected for a fibrillizing peptide. In contrast, the 1D-1H spectra of encapsulated Peptide 1 measured immediately after encapsulation and 4 days after encapsulation were essentially identical, with preservation of line width at 4 days, i.e., well within the time frame of most high-resolution NMR experiments. Encapsulation may provide a means to obtain high-resolution NMR data on unstable soluble oligomers of peptides implicated in amyloidoses such as Alzheimer's Disease and provide the first detailed structural information about these possibly cytotoxic species that have hitherto been inaccessible to analysis.

Helix-turn-helix peptides that form alpha-helical fibrils: turn sequences drive fibril structure.

Models of apolipoprotein A-I (apo A-I), the main protein of high-density lipoprotein, predict that it contains 10 amphiphilic, alpha-helical segments connected by turns. We synthesized four peptides with two identical 18-residue, amphiphilic, alpha-helical segments (Anantharamaiah, G. M., et al. (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I: (1) Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2, (2) Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2, (3) Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2, and (4) Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2. Surprisingly, peptides 1-3 formed fibrils after incubation (37 degrees C, 10 mM sodium phosphate, pH 7.60), but in contrast to beta-sheet amyloid fibrils, these did not bind thioflavin T and they induced a blue shift in the spectrum of Congo red. CD (peptides 1-3) and FTIR (peptides 1 and 2) of the fibrils showed significant alpha-helical character. Synchrotron X-ray fiber diffraction on a magnetically aligned sample of 1 confirmed the alpha-helical character in the fibrils and indicated that the helical axes are oriented perpendicular to the fibril axis. In contrast, peptide 4, containing two Gly residues but no Pro in the turn, formed only a small amount of nonfibrillar precipitate after prolonged incubation. Peptide 4P (peptide 4 with a Pro in place of the central Ala) and peptide 5, containing a PEG block in lieu of the central turn, were similar to peptide 4 in not forming fibrils, possibly because the region linking the helices was unstructured. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can drive the structure of fibrils.