Estimating the relative populations of 3(10)-helix and alpha-helix in Ala-rich peptides: a hydrogen exchange and high field NMR study.

Recent experimental and theoretical work suggests that alanine-rich peptides fold as a mixture of 3(10)-helix (i --> i + 3 hydrogen bonding) and alpha-helix (i --> i + 4 hydrogen bonding). In order to assess the relative proportions of the two conformers, NMR studies were performed on the 16 residue sequences: Ac-AAAAKAAAAKAAAAKA-NH2 (3K) and Ac-AMAAKAWAAKAAAARA-NH2 (MW). Hydrogen/deuterium-exchange kinetics measured for the first three amide protons of the 3K peptide indicate that the NH of Ala3 is partially protected from exchange. This result is consistent with the presence of an i --> i + 3 hydrogen bond between the carbonyl group of the acetyl blocking group and the NH group of Ala3. The MW peptide is a modified version of the 3K peptide, designed to increase alphaH signal dispersion. 1H NMR spectra of the MW peptide at 750 MHz reveal a series of intermediate range (NOEs) consistent with a mixture of 3(10)-helix and alpha-helix. The relative intensities of the alphaN(i,i + 3) and alphabeta(i,i + 3) (nuclear Overhauser enhancements) NOEs suggest that 3(10)-helix is present throughout the peptide, but with the greatest contribution at the termini. A model was developed to determine the relative contributions of 3(10)-helix and alpha-helix. Lower bounds for the population of 3(10)-helix are approximately 50% at the termini and 25% in the middle of the peptide. The greatest alpha-helical content is between the middle of the peptide and the N terminus.

Kinetics and mechanism of amyloid formation by the prion protein H1 peptide as determined by time-dependent ESR.

BACKGROUND: Peptides derived from three of four putative alpha-helical regions of the prion protein (PrP) form amyloid in solution. These peptides serve as models for amyloidogenesis and for understanding the alpha helix -->beta strand conformational change that is responsible for the development of disease. Kinetic studies of amyloid formation usually rely on the detection of fibrils. No study has yet explored the rate of monomer peptide uptake or the presence of nonfibrillar intermediate species. We present a new electron spin resonance (ESR) method for probing the kinetics of amyloid formation. A spin label was covalently attached to a highly amyloidogenic peptide and kinetic trials were monitored by ESR. RESULTS: Electron microscopy shows that the spin-labeled peptide forms amyloid, and ESR reveals the kinetic decay of free peptide monomer during amyloid formation. The combination of electron microscopy and ESR suggests that there are three kinetically relevant species: monomer peptide, amyloid and amorphous aggregate (peptide aggregates devoid of fibrils or other structures with long-range order). A rather surprising result is that amyloid formation requires the presence of this amorphous aggregate. This is particularly interesting because PrPSc, the form of PrP associated with scrapie, is often found as an aggregate and amyloid formation is not a necessary component of prion replication or pathogenesis. CONCLUSIONS: Kinetic analysis of the time-dependent data suggests a model whereby the amorphous aggregate has a previously unsuspected dual role: it releases monomer into solution and also provides initiation sites for fibril growth. These findings suggest that the beta-sheet-rich PrPSc may be stabilized by aggregation.

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHN alpha coupling constants.

Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar C alpha H-NH couplings (3JHN alpha) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the C alpha H region of 1H 2D NMR spectra. Quantitative determination of all possible 3JHN alpha values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson. 99, 552-560] have recently developed a method for determining 3JHN alpha from NOESY spectra. Because 3JHN alpha may be determined from 2D peaks outside of the C alpha H region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that 3JHN alpha can be obtained for every residue in the helical peptide Ac-(AAAAK)3A-NH2. The resulting 3JHN alpha profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins.

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrP(Sc)) can serve as a marker for TSE infectivity. STUDY DESIGN AND METHODS: Seven plasma protein-purification steps were performed after the plasma intermediates were spiked with TSE-infected material. Resulting fractions were analyzed for PrP(Sc) by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman-Kärber method. RESULTS: PrP(Sc) partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein-purification step. The detection ranges for PrP(Sc) and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP(Sc) and infectivity ranged from 1.0 to 6.0 log. CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP(Sc) and TSE infectivity. PrP(Sc) partitioning coincided with infectivity partitioning, which showed a close relationship between PrP(Sc) and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP(Sc) was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.