The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate~s! and trapping the folding intermediate~s! into enzymatically inactive, "aggregation-insensitive" state~s!. However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations~Ͼ1.5 M!, proline is shown to form loose, higher-order molecular aggregate~s!. The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, -hefutoxin1 and -hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that -hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any ␤؊sheets. Based on the presence of the functional diad (Tyr 5 /Lys 19 ) at a distance (6.0 ؎ 1.0 Å) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. -Hefutoxin 1 not only blocks the voltage-gated K ؉ -channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of -hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

In the present study, we demonstrate the thermal induced amyloid formation in a ␤-barrel protein, such as the acidic fibroblast growth factor from Notopthalmus viridescens (nFGF-1). Fibril formation in nFGF-1 is observed to occur maximally at 65°C. Electron microscope analysis of the thermal induced fibrils of nFGF-1 shows that they are filamentous with an average diameter of about 20 nm. X-ray diffraction analysis reveals that the thermal induced fibrils of nFGF-1 have a typical "cross-␤" structure with the ␤-strands perpendicular to the fibril axis. By using a variety of biophysical techniques including multidimensional NMR, we demonstrate that fibril formation involves the formation of a partially structured intermediate(s) in the thermal unfolding pathway of the protein (nFGF-1). Results of the anilino-8-napthalene sulfonate binding experiments indicate that fibril formation occurs due to the coalescence of the protein (in the intermediate state(s))through the solvent-exposed non-polar surface(s). In this study, we also demonstrate that organic osmolytes, such as proline, can efficiently prevent the thermal induced amyloid formation in nFGF-1. Proline is found to stabilize the native conformation of the protein. The addition, proline is observed to increase the cooperativity of the unfolding (native 7 denatured) reaction and consequently decrease the population of the "sticky" thermal equilibrium intermediate(s) responsible for the fibril formation.

between the tautomers of methyl-9-hydroxyphenalen-1 -one was found to be 154 cm"1 234, in good agreement with the results from laser-excited fluorescence and fluorescence-excitation spectroscopy. 13,15 The time evolution of the probability density of the initially localized state function demonstrates that, for the symmetric case, the proton or deuteron reaches the other well at the frequencies of their respective gerade-ungerade splittings. For the asymmetric case, three-tenths of a proton, but less than three-hundredths of a deuteron, reaches the other well with each oscillation of frequency equal to the energy difference between the two lowest eigenstates, 5.03 X 1013 and 4.52 X 1013 s, respectively.Although the introduction of the methyl substituent destroys the symmetry of the double-mininum potential, the rate is fast because the barrier, the interminimal distance, and the difference in energy between the two tautomers are small for this nearly symmetric potential energy profile. This explains the NMR spectra interpreted as showing that methylnaphthazarin does not have proton exchange, but methyl-9-hydroxyphenalen-1 -one does.Tunneling in near symmetric cases deserves special attention. In symmetric cases, one can calculate tunneling from the energy difference between the gerade-ungerade pair. This is so because the proton will spend equal time in each well, and tunnelling will depend on the frequency with which the proton oscillates between the wells. When the profile is asymmetric, the fraction of the proton that leaks to the other well decreases as the energy difference between the minima increases. It has been found26 that this fraction depends on the ratio of the energy difference between the minima and the tunneling splitting in the corresponding symmetric case. In the present study the fraction of the proton that leaks to the other well is reduced from unity in the symmetric case to one-third in this nearly symmetric case, while the frequency with which the maximum leakage occurs increases by 60%.Acknowledgment. We thank Professor L. M. Jackman of the Pennsylvania State University for stimulating our interest in 9-hydroxyphenalen-1-one, Professor J. H. Busch of Villanova University for his helpful discussions, Timothy Ay of the Villanova University Computer Center, Dr. Eugene M. Fluder from Merck Sharp and Dohme, and the staff of the Villanova University Computer Center for their help with the various programs.

Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

The refolding kinetics of the 140-residue, all ␤-sheet, human fibroblast growth factor (hFGF-1) is studied using a variety of biophysical techniques such as stoppedflow fluorescence, stopped-flow circular dichroism, and quenched-flow hydrogen exchange in conjunction with multidimensional NMR spectroscopy. Urea-induced unfolding of hFGF-1 under equilibrium conditions reveals that the protein folds via a two-state (native 7 unfolded) mechanism without the accumulation of stable intermediates. However, measurement of the unfolding and refolding rates in various concentrations of urea shows that the refolding of hFGF-1 proceeds through accumulation of kinetic intermediates. Results of the quenchedflow hydrogen exchange experiments reveal that the hydrogen bonds linking the N-and C-terminal ends are the first to form during the refolding of hFGF-1. The basic ␤-trefoil framework is provided by the simultaneous formation of ␤-strands I, IV, IX, and X. The other ␤-strands comprising the ␤-barrel structure of hFGF-1 are formed relatively slowly with time constants ranging from 4 to 13 s.

The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all ␤-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy.

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