Intermediacy of Poly(l-proline) II and β-Strand Conformations in Poly(l-lysine) β-Sheet Formation Probed by Temperature-Jump/UV Resonance Raman Spectroscopy

Abstract

Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperaturejump (T-jump) was used to investigate early steps in the temperature induced α-helix to β-sheet conformational transition of poly(L-lysine) (poly(K)). Excitation at 197 nm from a tunable frequency-quadrupled Ti:Sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although unionized poly(K) (pH 11.6) is mainly α-helical below 30°C, there is a detectable fraction (∼15 %) of unfolded polypeptide, which is mainly in the polyproline II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be β-strand. Above 30°C unionized poly(K) forms β-sheet at a rate (minutes) which increases with increasing temperature. A 22 -44°C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/β-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, ∼ 2 % at the 3 μs time limit of the experiment. The rise time of the PPII signals is ∼250 ns, consistent with melting of short α-helical segments. A model is proposed in which the melted PPII segments interconvert with β-strand conformation, whose association through inter-strand H-bonding nucleates the formation of β-sheet. The intrinsic propensity for β-strand formation could be a determinant of β-sheet induction time, with implications for the onset of amyloid diseases. KeywordsUVRR; resonance Raman; protein folding; poly(L-lysine); PPII; β-strand; α-helix; β-sheet; temperature-jump; T-jump Several degenerative diseases, including Alzheimer's, Huntington's, Parkinson's, and transmissible spongiform encephalopathies (TSE), are associated with misfolding and aggregation of proteins (1). The misfolded proteins form fibrillous aggregates, termed amyloid fibrils, which have a cross β-sheet structure in which the polypeptide backbone is perpendicular to the fibril axis(2). This allows the fibril to grow lengthwise with the addition of subsequent misfolded protein molecules. It is interesting to note that conditions which promote partial folding of proteins, appear to promote fibril formation in vitro (3)(4)(5). † This work was supported by NIH Grants R01 GM 025158 to T.G.S. and NIH F32 AG 022770 to R.D.J. * To whom correspondence should be addressed: Princeton University, Department of Chemistry, Princeton, NJ, 08544. Telephone: (609) Certain proteins with abundant α-helical structure, including the prion protein (PrP C ) and apomyoglobin, convert to β-sheet rich structures under denaturing conditions (4-6). What is equally provocative is the correlation between metastable α-helices and β-sheet amyloid formation. The β-amyloid (Aβ) peptide involved in Alzheimer's disease and its constituent fragments contain no stable secondary structure in aqueous solutions (7,8), but when the nascent helic...

The effects of shear flow on protein structure and function

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“…86 In addition, abrupt conformational transitions are not expected in simple shear flow since the occurrence of extensional strain, hence polymer stretching, is random. It is hypothesized that at very high strain rates, where polymer chains spend a relative short time (residence time) in an extensional flow field, no unfolding or incomplete chain extensions may occur.…”

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