A new algorithm, called convex constraint analysis, has been developed to deduce the chiral contribution of the common secondary structures directly from experimental CD curves of a large number of proteins. The analysis is based on CD data reported by Yang, J.T., Wu, C.-S.C. and Martinez, H.M. [Methods Enzymol., 130, 208-269 (1986)]. Application of the decomposition algorithm for simulated protein data sets resulted in component spectra [B (lambda, i)] identical to the originals and weights [C (i, k)] with excellent Pearson correlation coefficients (R) [Chang, C.T., Wu, C.-S.C. and Yang, J.T. (1978) Anal. Biochem., 91, 12-31]. Test runs were performed on sets of simulated protein spectra created by the Monte Carlo technique using poly-L-lysine-based pure component spectra. The significant correlational coefficients (R greater than 0.9) demonstrated the high power of the algorithm. The algorithm, applied to globular protein data, independent of X-ray data, revealed that the CD spectrum of a given protein is composed of at least four independent sources of chirality. Three of the computed component curves show remarkable resemblance to the CD spectra of known protein secondary structures. This approach yields a significant improvement in secondary structural evaluations when compared with previous methods, as compared with X-ray data, and yields a realistic set of pure component spectra. The new method is a useful tool not only in analyzing CD spectra of globular proteins but also has the potential for the analysis of integral membrane proteins.

The 0-turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the a-helix and 0-pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of @-turns. Recently, it has been reported (Hollosi, M., Kover, K.E., Holly, S., Radics, L. [9772][9773][9774][9775][9776][9777][9778][9779][9780][9781][9782][9783][9784]) that some pseudohexapeptides (e.g., the cyclo[(6)Ava-Gly-Pro-Aaa-Gly] where Aaa = Ser, Ser(O'Bu), or Gly) in many solvents adopt a conformational mixture of type I and the type I1 &turns, although the X-ray-determined conformation was an ideal type I 0-turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above &turn models has an influence on their conformational properties.Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type I1 @-turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnady, G., Hollosi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669-679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 0-turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major 0-turns by CD analysis. These results can now be used to enhance the conformational determination of globular proteins on the basis of their CD spectra.

Circular dichroism (CD) and1 H‐{1H}NOE spectra were obtained for Piv‐Pro‐Ser‐NHCH3(1),[Piv‐(CH3)3‐C‐CO], Boc‐Pro‐Ser‐NHCH3 (2) and Boc‐Val‐Ser‐NHCH3 (3), to determine the solution conformation of these p‐turn models. In the crystal, 1 and 3 adopt an ideal type I β‐turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc‐Pro tert‐amide bond. The predominance of a β‐turn conformation in solution was suggested for models 1‐3 on the basis of 1H‐{1H}NOE data. In a nonpolar solvent the prevailing trans rotamer form (>80%) of 2 has a β‐turn conformation according to heteronuclear NOE measurement. Positive 1H‐{1H} NOEs were detected between the Hα(Pro)/NH(Ser), Hα(Ser)/NH(Ser) and NH(NHCH33)/HN(Ser) protons in the trans Boc‐Pro rotamer form of 2 at ‐20° in CDCl3. Similar positive homonuclear NOE enhancements were also observed on the appropriate proton signals in other models, such as Boc‐Val‐Ser‐NHCH3 (3). Boc‐Val‐D‐Ser‐NHCH3 (4) and Boc‐Pro‐D‐Ser‐NHCH3 (5), in various solvents. The 1H‐ {1H)NOE experiments carried out in CD3CN clearly showed that besides the type I (or III) β‐turn structure, one of the main conformations of models 1‐5 is close to the type II β‐turn backbone geometry in a nonpolar solvent. Unexpectedly, the conformational mixture of models 1‐3 were characterized by class C (helix‐like) CD spectra, although class C spectra are generally only correlated with the type I β‐turn conformation. These acyclic models are the first carefully investigated examples of ‐L‐L‐ triamide systems, containing a significant amount of a type II β‐turn, as well as the type I p‐turn and, however, yielding a class C circular dichroism spectra. The CD spectra recorded for 3 and 4 in acetonitrile were ‘calibrated’ using the 1H‐{1H}NOE data. Such a “calibration”, as well as the semi‐quantitative CD and NMR comprehensive analyses, demonstrated that class C, class B, as well as class C’ CD spectra may be obtained from the linear combination of the same two‐component spectra, with different conformational weights. Therefore, it is suggested that the extraction of the conformational components of such models, simply on the basis of their CD spectra, must be made with caution.

NF-M13 [H-(Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly)-OH], NF-M17 [H-(Glu-Glu-Lys-Gly-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) -OH], and their phosphorylated derivatives, representing the C-terminal phosphorylation domain of the neurofilament protein midsize subunit, have four possible binding sites for metal ions: the COO- group of glutamate, the OH group of the serine residue, the PO3H- group of phosphoserine (when present), and the COO- at the terminus of the peptide chain. The CD titration of the phosphorylated neurofilament fragments with Al3+ and Ca2+ yielded a significant conformational change that resulted in conformations containing high beta-pleated-sheet contents, which precipitate on standing (intermolecular complex). Al3+ binding to the unphosphorylated NF-M13 and NF-M17 did not exhibit this behavior. Several alanine analogues of the parent NF-M17 peptide were synthesized in order to determine the relationship between metal ions and possible binding sites. CD titration of analogues with Ca2+ indicated that the critical residues of NF-M17 for Ca(2+)-induced conformational changes, from random to beta-pleated sheet, are the N-terminal serine or both phosphorylated serines. Al(3+)-induced conformational changes suggest that the critical sites of NF-M17 yielding the beta-pleated-sheet structure are the four glutamates or phosphorylated serines, especially the C-terminal SerP. On the basis of the titration data, it is very likely that analogues with a serine in position 11 form a stable intramolecular complex with Al3+ that, however, does not result in the adoption of the beta-conformation. Back-titration with citric acid fails to reverse the Al(3+)-induced conformational changes of the phosphorylated peptides. The above results, especially the possible formation of intramolecular and intermolecular Al3+ complexes, may have relevance to the molecular mechanism, through which the neurotoxin Al3+ gives rise to the formation of neurofilament tangles.

Plaques are one of the two lesions found in the brain of patients with Alzheimer disease. Using a synthetic peptide corresponding to rat ,B-amyloid-(1-42) (j3A4), circular dichroism (CD) analyses were performed to examine the effect of Na4SiO4 on the conformational state produced by Al3+. A previous study on fragments of neuronal proteins involved in tangle formation had shown a conformational transition from a 8-pleated sheet to a soluble random coil upon addition of Na4SiO4. In the present study, CD measurements showed that the f3-pleated sheet conformation of f3A4 induced by Al3+ was reversed to the random coil soluble form by the addition of Na4SiO4. The tight binding of Al3+ provides the mechanism for this transition. These results provide insight into the role of aluminum in the Alzheimer diseased brain and suggests the investigation of the use of silicates as a therapeutic agent.Alzheimer disease (AD) will probably become the predominant social-medical-economic problem in the next century. An estimated 4 million Americans now have the disease, and this number is expected to grow to >10 million within the next decade (1). The cause of AD is not known, and treatments are only mildly effective at slowing the degeneration process (2). The signs of AD, obtained upon autopsy of the diseased brain, reveal two phenomena: amyloid plaques and neurofibrillary tangles (3). The amyloid plaques are composed primarily of a 39-to 42-amino acid fragment (13A4) from the amyloid precursor protein (4). Tangles contain abnormally phosphorylated tau protein that combine to form paired helical filaments within the neuron (5). In addition to the protein components of plaques and tangles, some researchers have detected aluminum (6, 7) and aluminosilicates (8) in these lesions, although the evidence for their presence is apparently controversial (9).Aluminum is known to have toxic effects (10) on many organisms, including man. Aluminum is an associated risk factor in amyotrophic lateral sclerosis, Parkinsonismdementia of Guam, and AD (11,12).Aluminum is the third most abundant element in the earth's crust, found primarily as insoluble aluminosilicates and oxides. However, it is soluble as aluminum hydroxide and aluminum hydrates at acidic pH values (13). This has become a biologic concern as the world's water supplies become more acidic due to acid rain. For (18,19). The formation of 13-pleated sheets could be reversed by the addition of silicates (20).The current paper is a study of the ,3A4 peptide (residues 1-42 of the ,B-amyloid precursor protein), whose prominent role in the progression of AD has been frequently stressed and has been reviewed extensively (21-23). Several structural studies on 13A4 or its fragments have been reported (24)(25)(26)(27)(28). CD studies illustrated the requirements for 13-sheet filament formation (29). Solid-state Fourier-transform infrared spectroscopy showed that the secondary structure consisted of a 13-turn flanked by two strands of antiparallel 1-pleated sheet (30). In aqueous tri...

The three-dimensional structure of a protein is the assembly of diferent scna strutural elements, such as a-helices, 3-pleated sheets, and -turns.

CD spectroscopic studies on protected peptides containing lysine and serine, or phosphoserine, and on serine-containing fragments of the neurofilament protein midsized subunit, both in the unphosphorylated and phosphorylated form, are reported. The introduction of the phosphoryl group was not found to have a significant spectral effect in aqueous solution. In trifluoroethanol (TFE), spectral shifts toward unordered (type U) spectra or the appearance of distorted spectra likely reflect the adoption of aperiodic polypeptide conformations due to salt bridge(s) between negatively charged phosphoserine and positive lysine side-chain groups. A turn-stabilizing effect of phosphorylation was also observed. CD-monitored titration experiments in TFE revealed a high conformational sensitivity of phosphopeptides toward Ca2+ ions. The appearance of the unordered spectra or spectral shifts were the sign of a bulk disordering effect of Ca2+ ions. Spectra with specific spectroscopic features reflect the formation of Ca2+ complexes and the adoption of ordered unique backbone conformations. When ordered structures were obtained on addition of Ca2+ ions, the observed CD curves showed a resemblance to the spectrum of beta-pleated sheets. This may originate from chain extension and the formation of beta-pleated sheet segments fixed by Ca2+ bridges between PO3H-1 groups of adjacent peptide chains. The data clearly show that the effect of the Ca2+ ions is highly specific: the sequence, chain length, presence and distribution of charged side-chain groups, degree and site of phosphorylation, and environmental factors appear to be determining in the process of chain extension or beta-sheet formation.