(PDF) A Designed System for Assessing How Sequence Affects alpha to beta Conformational Transitions in Proteins

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 12, Issue of March 22, pp. 10150 –10155, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Designed System for Assessing How Sequence Affects ␣ to ␤ Conformational Transitions in Proteins* Received for publication, August 10, 2001, and in revised form, December 11, 2001 Published, JBC Papers in Press, December 21, 2001, DOI 10.1074/jbc.M107663200 Barbara Ciani‡§, E. Gail Hutchinson¶, Richard B. Sessions储, and Derek N. Woolfson‡** From the ‡Centre for Biomolecular Design and Drug Development, The School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom, the ¶Division of Cell and Molecular Biology, of Biochemistry School of Animal and Microbial Sciences, University of Reading, Whiteknights, P. O. Box 228, Reading RG6 6AJ, United Kingdom, and 储The Department of Biochemistry, The School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom The role of amino acid sequence in conformational the diseases collectively known as amyloidoses (3–5). There- switching observed in prions and proteins associated fore, the elucidation of the underlying molecular principles for with amyloid diseases is not well understood. To study ␣ the transformation of soluble proteins into amyloid has poten- to ␤ conformational transitions, we designed a series of tial for understanding and tackling these diseases. peptides with structural duality; namely, peptides with A diverse set of peptides and proteins form amyloid (6, 7). sequence features of both an ␣ⴚhelical leucine zipper This set is not limited to peptides and proteins that form Downloaded from http://www.jbc.org/ by guest on January 31, 2016 and a ␤ⴚhairpin. The parent peptide, Templateⴚ␣, was amyloid deposits in vivo and are associated with the various designed to be a canonical leucine-zipper motif and was diseases (8, 9), and even non-natural, designed peptides and confirmed as such using circular dichroism spectros- proteins can assume amyloid-like structures (10 –12). In these copy and analytical ultracentrifugation. To introduce cases, the structural changes vary from slow conversions of ␤ⴚstructure character into the peptide, glutamine resi- random-coil peptides to gross structural changes of larger, na- dues at sites away from the leucine-zipper dimer inter- face were replaced by threonine to give Templateⴚ␣T. tively folded, globular structures brought about by destabiliza- Unlike the parent peptide, Templateⴚ␣T underwent a tion of the native state. Furthermore, for the latter, there are heat-inducible switch to ␤ⴚstructure, which reversibly no clear themes in the types of native state that undergo formed gels containing amyloid-like fibrils. In contrast transitions to amyloid: all-␣-helical proteins can be trans- to certain other natural proteins where destabilization formed into amyloid (13), as can all-␤-structures (14) and struc- of the native states facilitate transitions to amyloid, de- tural types between these extremes (6, 7). Nonetheless, amy- stabilization of the leucine-zipper form of Templateⴚ␣T loid and amyloid-like structures have common cores based on did not promote a transformation. Cross-linking the ter- ␤-structure (15, 16): fibrils are assembled from protofilaments mini of the peptides compatible with the alternative in which ␤-strands are aligned perpendicular to the long fiber ␤ⴚhairpin design, however, did promote the change. axis. Thus, an increasingly adopted view is that the ability to Furthermore, despite screening various conditions, form amyloid is largely a general property of the polypeptide only the internally cross-linked form of the parent, Tem- backbone (9, 17). However, the role, if any, of protein sequence plateⴚ␣, peptide formed amyloid-like fibrils. These find- in amyloid fibril formation is not clear. ings demonstrate that, in addition to general properties A number of studies indicate that sequence, and therefore of the polypeptide backbone, specific residue place- amino acid side chains, do influence the formation of amyloid ments that favor ␤ⴚstructure promote amyloid (10, 18 –22). However, the question is whether sequence formation. changes simply affect the relative stabilities of the various folded, partly folded, and unfolded states of the subject proteins and hence their propensity to aggregate (18, 20, 21, 23); in this Gross conformational transitions (or switches) in proteins sense, the role of sequence in the formation of the amyloid are increasingly coming to light. Broadly speaking these may structure itself may be regarded as passive. Alternatively, se- be classed into two types: those that have evolved to tailor or quence may take a more active role in promoting ␤-structured elicit specific normal protein functions, and those that lead to elements in the unfolded states or within the amyloid-fibril aggregated forms that render proteins defunct or even patho- structures themselves (10, 19, 22). genic. Examples of the first group include the large structural We are interested in addressing a specific issue in protein changes of certain viral-coat proteins that accompany virus- conformation switching, namely, how sequence brings about host membrane interactions (1). The other type of structural and influences the rather extreme ␣ to ␤ structural transitions change is associated with the prions and proteins that form in proteins. To do this, we set out to design a peptide with a amyloid (2, 3). structural conflict; namely, with sequence features compatible It is accepted that misfolding of peptides and proteins cause with both ␣-helical and ␤-hairpin structures. Such a system the fibrillar aggregates known as amyloid, which characterize would allow us to assess the role of specific side chain place- ments in effecting the conversion between the two structures; * The costs of publication of this article were defrayed in part by the in addition, if the ␤-structured form acted as precursor for payment of page charges. This article must therefore be hereby marked amyloid formation, such a system may provide insight into the “advertisement” in accordance with 18 U.S.C. Section 1734 solely to mechanism(s) of conversion to amyloid-like structures. indicate this fact. § Supported by a University of Sussex D.Phil. studentship. Others have engineered peptides and proteins with struc- ** To whom correspondence should be addressed. Fax: 44-0-1273- tural conflicts. Minor and Kim (24) show that an 11-residue 678433; Tel.: 44-0-1273-678214; E-mail:

. peptide sequence can be accommodated at structurally distinct 10150 This paper is available on line at http://www.jbc.org A Designed ␣⫺ to ⫺␤-peptide Switch 10151 FIG. 1. Design principles and designed peptide sequences. A and B, schematic representations of a heptad sequence repeat (abcdefg) configured onto an ␣-helical wheel with 3.5-residues per turn, and a ␤-hairpin, respectively. In the latter the dashed lines indicate the intended inter-strand hydrogen-bonded sites; that is, positions where the backbones of the residues paired across the ␤-hairpin should hydrogen bond. C, designed peptide sequences. Key: standard one-letter codes are used for the amino acids; Ac, acetyl (CH3.C.O.-); Am, amidated C terminus (-NH2). Color is used to highlight residues designed to interact in both structures: green, hydrophobic residues; dark blue, positively charged lysines; red, negatively charged glutamates. The f positions where the Gln to Thr substitutions were made are colored light blue. Downloaded from http://www.jbc.org/ by guest on January 31, 2016 sites within the same protein fold; in this case, overall tertiary peptides). All data were collected in 1-mm quartz cuvettes. Data points context overrides local sequence and secondary structure pref- for CD spectra were recorded at 1-nm intervals using a 1-nm bandwidth and 4 –16-s response times. After baseline correction, ellipticities in erences. At the level of a whole protein, Dalal and Regan (25) mdeg were converted to molar ellipticities (deg cm2 dmol res⫺1) by have met the Paracelsus Challenge and succeeded in transmut- normalizing for the concentration of peptide bonds. Data points for the ing a mixed ␣/␤ protein fold into an all ␣⫺helical fold by thermal unfolding curves were recorded through 1 °C min⫺1 ramps altering only ⬇50% of the sequence. In addition, several groups using a 2-nm bandwidth, averaging the signal for 16 s at 1 °C intervals. have succeeded either serendipitously, or with reasoned de- Analytical Ultracentrifugation—Sedimentation equilibrium experi- signs to construct peptide sequences that do switch conforma- ments were conducted at 5 °C in a Beckman Optima XL-I analytical ultracentrifuge fitted with an An-60 Ti rotor. A ⬇100-␮l sample of tional state (11, 12, 26 –29). Template-␣ (100 ␮M) in standard buffer containing 100 mM sodium To elucidate specific sequence features of natural proteins chloride was used. The sample was equilibrated for ⬇48 h at rotor that drive ␣ to ␤ secondary structure switching, we designed speeds of 40,000, 50,000, and 60,000 rpm. Sedimentation curves were and characterized a series of peptides in which positive design measured by absorbance at 240 nm (the E240 for Template-␣ was cal- features for a dimeric, ␣-helical coiled coil and a ␤-hairpin were culated as 2240 M⫺1 cm⫺1). The resulting data sets were fitted simul- superimposed in a short sequence. The parent peptide, Tem- taneously using routines in the Beckman Optima XL-A/XL-I data anal- ysis software (version 4.0). Two fitting models were used: the first plate-␣, was confirmed as a stable, cooperatively folded, di- assumed a single ideal species; the second assumed a monomer-dimer meric, helical structure consistent with the designed leucine equilibrium and fixed monomer molecular weight. The molecular zipper. As expected, mutation of three exterior Gln residues to weight and partial specific volume of the peptide were calculated from Thr reduced the stability of this folded state. Surprisingly, the amino acid sequence as 3118 and 0.755, respectively. The viscosity however, thermal unfolding of the mutant was accompanied by of the buffer at 5 °C was taken to be 1.008 mg ml⫺1. conversion to ␤-structure on a time scale of tens of minutes. In Thioflavine T Binding—Emission fluorescence spectra of thioflavine T (10 ␮M) with ⬇10 ␮M peptide added were recorded between 480 and this state the samples gelled and were shown to contain fibrils 600 nm with an excitation wavelength of 435 nm using a Varian Eclipse with tinctorial properties and the morphology of amyloid. spectrofluorimeter and 1-cm quartz cuvettes. A scan rate of 600 nm Small sequence changes were made to probe the basis of the min⫺1 and data interval of 1 nm was used throughout. conversion to amyloid. We conclude that straight destabiliza- Electron Microscopy—Droplets of peptide solution were applied to tion of the coiled-coil structure does not necessarily foster the carbon-coated copper specimen grids and dried with filter paper before change, but alterations geared to favor the alternative ␤-con- negative staining with 2% phosphotungstic acid at pH 7. Grids were examined in a Hitachi 7100 TEM at 100 kV and digital images were formation do promote the structural switch. acquired with a (800 ⫻ 1200 pixel) charge-coupled device camera. EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION Peptide Synthesis—Peptides were made on a Pioneer Peptide Syn- Design Principles and Characterization of Template-␣—The thesis System (Perseptive Biosystems) using standard Fmoc chemistry. Peptides were purified by reverse-phase high performance liquid chro- starting point for our study was a designed canonical ␣-helical matography and their identities confirmed by matrix-assisted laser leucine-zipper sequence (Template-␣). Using established rules desorption ionization-time of flight mass spectrometry. Purified pep- for coiled-coil assembly (30 –33), Template-␣ was designed to tides were stored at pH 2, ⫺20 °C in 7 mM DTT.1 Oxidized peptides were form a leucine zipper (i.e. a dimeric, parallel coiled coil, Fig. prepared as follows: peptides (at ⱕ100 ␮M) were agitated at room 1A): the combination of Val at a and Leu at d sites of the heptad temperature overnight in 100 mM Tris (pH 8.5), 20% Me2SO, 2 M repeat was used to direct dimer formation; Lys at one a position guanidine hydrochloride. Oxidized peptides were stored at pH 2, ⫺20 °C. and the juxtaposition of Lys and Glu at g and e, respectively, Circular Dichroism Spectroscopy—Circular dichroism measure- were placed to ensure parallel dimers; the b and c positions ments were made on a JASCO J-715 spectropolarimeter fitted with a were filled with helix-promoting Ala residues and the outer f Peltier temperature controller. Unless otherwise stated, the sample sites were made polar Gln. The heptad repeat was flanked by stocks were diluted into a standard buffer of 25 mM potassium phos- Cys residues, which could be oxidized to an intramolecular phate (pH 7) containing 1 mM DTT (DTT was omitted for the oxidized disulfide bond to favor the alternative ␤-hairpin conformation (Fig. 1B) as required. An N-terminal Tyr-Gly tag was added to 1 The abbreviation used is: DTT, dithiothreitol. allow peptide concentrations to be determined (34). The N and C 10152 A Designed ␣⫺ to ⫺␤-peptide Switch rium model gave a dissociation constant of 66 ␮M (with 95% confidence limits of 49 and 91 ␮M). These data are thus con- sistent with the CD measurements, which indicated near, but not complete folding at 5 °C and 100 ␮M peptide concentrations. In summary, the CD and equilibrium sedimentation data for Template-␣ are comparable with those collected on other de- signed and natural leucine zippers under similar conditions (35) and are, thus, consistent with the peptide forming a di- meric helical structure as designed. Promoting a Conformational Switch in the Template-␣ Sys- tem—As a first step to introduce ␤-structure character into the designed system, the three Gln residues at the f sites of the heptad repeat of Template-␣ were replaced by Thr to give Template-␣T (Fig. 1C). The f sites lie away from and, so, should not compromise the leucine-zipper interface (Fig. 1A). As Thr has a lower ␣-helix propensity and a higher ␤-sheet propensity than Gln (36, 37) Template-␣T was expected to form a leucine zipper, but with a reduced stability compared with Template-␣. This was observed (compare Figs. 2, A and B, and 3, A and B). However, the thermal unfolding of Template-␣T was less re- versible; that is, spectra recorded after returning to the start- ing temperature showed a lower helicity than was measured at Downloaded from http://www.jbc.org/ by guest on January 31, 2016 the start of the experiment (Fig. 3A). When thermal unfolding was repeated at the higher peptide concentration of 300 ␮M, Template-␣ again unfolded with a normal, reversible, sigmoi- dal transition (Fig. 2, A and B). By contrast, the unfolding of Template-␣T showed an inflection above 60 °C, which sug- gested some refolding (Fig. 3B). Spectra recorded after cooling this sample were wholly different from anything recorded pre- viously: the minima at 208 and 222 nm were absent and re- placed by a single minimum at 218 nm, indicative of ␤-struc- ture (Fig. 3A). It was possible to follow the transition of Template-␣T from a near-unfolded conformation to a ␤-confor- mation directly on the time scale of minutes by maintaining a freshly prepared 300 ␮M sample of the peptide at 70 °C (Fig. FIG. 2. Experimental characterization of the parent peptide, Template-␣. A, circular dichroism (CD) spectra recorded at 5 °C in 25 3C), i.e. just beyond the inflection point in the unfolding curve. mM phosphate buffer at pH 7 containing 1 mM DTT. Solid lines show Coupled with these structural changes the samples of Tem- spectra recorded before thermal unfolding experiments and broken lines plate-␣T gelled. To test if this process could be reversed the gel are for those recorded after thermal unfolding. Solid circles are for data recorded at 100 ␮M peptide and open circles are for 300 ␮M samples. B, was re-suspended in a 25 mM phosphoric acid buffer adjusted to thermal unfolding curves followed by measuring the CD signal at 222 pH 2; the rationale being that peptides, proteins, and protein nm as a function of temperature; the key is same as for part A. C, complexes can be acid denatured, presumably because at low sedimentation-equilibrium analysis. The open circles are experimental pH polypeptides carry net positive charges, which repel in the data collected at 40,000 rpm for a 100 ␮M sample at 5 °C in 25 mM phosphate buffer at pH 7 containing 1 mM DTT and 100 mM NaCl. The folded and/or associated states. The CD spectrum recorded for lines are calculated curves for ideal monomer (dashed line), dimer (solid the Template-␣T gels recorded immediately after re-suspen- line), and trimer (dotted line) states of Template-␣. sion indicated ␤-structure (Fig. 3D). Interestingly, however, with time the CD spectrum of this sample reverted to that for termini were acetylated and amidated, respectively. The full a partly folded ␣-helix (Fig. 3D). sequences of Template-␣ and its derivatives are given in Fig. 1C. Template-␣T Formed Amyloid-like Fibrils—Switches to The coiled-coil conformation of Template-␣, at 100 ␮M con- ␤-structure accompanied by gel formation are indicative of centration, 25 mM potassium phosphate, 1 mM DTT (pH 7), was amyloid-like fibrils. Therefore, we tested the gelled peptide confirmed experimentally using a combination of circular di- samples for tinctorial properties characteristic of amyloid. The chroism spectroscopy and analytical ultracentrifugation (Fig. fluorescence of thioflavine T (38) was compared in the presence 2). The CD spectrum of Template-␣ at 5 °C had minima at 208 of Template-␣T in its ␣-helical and ␤-structured states. The and 222 nm characteristic of ␣-helical structure (Fig. 2A). From fluorescence intensity at 480 nm for the latter was ⬇2.5 times the intensity of the signal at 222 nm, we estimated that Tem- greater (Fig. 4A). This was evidence for the presence of amy- plate-␣ contained ⬇70% helix (35). The thermal unfolding of loid-like fibrils in the heat-treated, 300 ␮M Template-␣T sam- Template-␣ was concentration dependent as expected for an ple. To confirm this we prepared samples of this state for oligomerizing system (Fig. 2B). Furthermore, the unfolding electron microscopy. The resulting images revealed filamen- transitions were almost completely reversible (Fig. 2A). tous, 25–30-nm bundles of extended, non-branching fibrils (Fig. Sedimentation equilibrium studies of Template-␣ indicated a 4, B and C). The component fibrils were twisted with a similar monomer-dimer equilibrium (Fig. 2C), consistent with the CD periodicity and, on average, had a diameter of ⬇10 nm. These data and the design. For example, the data did fit an analysis features are all consistent with amyloid formation. that assumed a single ideal species, but returned a Mr of 5273 Manipulating the ␣ to ␤ Transition in the Template-␣ Sys- (with 95% confidence limits of 5036 and 5503), i.e. between the tem—Having established a system that underwent an ␣ to ␤ molecular weights expected for monomer (3118) and dimer. transition with the added curiosity that the ␤-structured state More detailed analysis assuming a monomer-dimer equilib- formed amyloid, we set out to examine how small, specific and A Designed ␣⫺ to ⫺␤-peptide Switch 10153 FIG. 3. Structural transitions in Template-␣T. A, CD spectra recorded at 5 °C in 25 mM phosphate buffer, 1 mM DTT (pH 7). Solid lines are spectra re- corded before thermal unfolding experi- ments and broken lines are for those re- corded after thermal unfolding. Solid circles are for data recorded at 100 ␮M peptide and open circles are for 300 ␮M samples. B, thermal unfolding curves; the key is same as for part A. C, time-depend- ent change in the CD spectra of a 300 ␮M sample at 70 °C. Open squares, t ⫽ 0; solid squares, t ⫽ 120 min; intervening lines were recorded at 10, 20, 30, 40, 50, and 60 min. D, CD spectra of “gels” formed by heat-treated 300 ␮M Template- ␣T. The gels were re-suspended in 25 mM phosphate (pH 2) and spectra recorded immediately (solid line) and after 5 days (broken line). peptides (40).2 Thus, Template-␣T was re-synthesized with free Downloaded from http://www.jbc.org/ by guest on January 31, 2016 N and C termini to give Template-␣Tu (Fig. 1C). This had the desired effect of destabilizing the leucine-zipper state; indeed, the peptide was random coil and did not form appreciable helical structure over a wide range of concentrations. It is not clear how uncapping the peptide might affect amyloid forma- tion itself. Nevertheless, Template-␣Tu could be converted to a ␤-structured state. However, this still required elevated tem- peratures and, in fact, the transition was more difficult to effect than with Template-␣T: under the conditions used to convert Template-␣T (300 ␮M, 70 °C), Template-␣Tu did not transform over a 1-h time scale, but a transition was observed within 1 h at 80 °C. Fibrils were observed for these samples by electron microscopy. With Template-␣Tu the formation of amyloid was assisted by salt; 0.5 M KF lowered the temperature required for the con- version described above to 70 °C, i.e. to that required for the capped peptide in the absence of salt. Curiously, however, salt also stabilized the low-temperature ␣-helical state of the pep- tide increasing the ␣-helical content from ⬇33 to 50% at 300 ␮M peptide. Thus, again for our system, there was no correlation between destabilization of the native state and easing fibrillo- genesis. Salt also accelerated the conversion of Template-␣T at 70 °C to the ␤-structured state; the CD-monitored transitions in 0, 0.5, and 1 M KF were complete in ⬇60, 30, and 10 min, respectively. Promoting a ␤-Hairpin Structured Intermediate?—Summa- rizing the above, simply destabilizing the leucine-zipper con- formation of Template-␣T did not facilitate the ␣ to ␤ transi- tion. Others have used ␤-hairpins to build a high-resolution structure for an A␤ amyloid fibril (15), and implicated their involvement in amyloid-like fibril formation by a peptide from OspA (41). Therefore, our next step was to determine if the transition was affected by increasing the ␤-hairpin propensity FIG. 4. Amyloid-like fibrils formed by Template-␣T. A, fluores- of Template-␣ (Fig. 1B). Although by no means an ideal ␤-hair- cence spectra of solution of 10 ␮M thioflavine T recorded with Tem- pin sequence, Template-␣ was designed to be compatible with plate-␣T in the ␣-helical state (solid line) and the heat-treated ␤-struc- this conformation. The key features were the Cys residues tured state (broken line). B and C, transmission electron micrographs of flanking the central heptad-based sequence. In our design stained, heat-treated Template-␣T at ⫻15,000 and 60,000 magnifica- tion, respectively. logic, oxidation of these to an intramolecular disulfide link would bring the termini of the peptide together. This should rational sequence changes influenced the transition. For in- simultaneously promote the ␤-hairpin and destabilize the stance, could the transition be eased and made to occur at lower leucine zipper. The design principles for the ␤-hairpin follow temperatures by destabilizing the native coiled-coil conforma- from an understanding of amino acid pairings in anti-parallel tion of Template-␣T? Capping the ends of free-standing ␣-hel- ␤-sheets (42, 43). On this basis, the Cys-Cys pair should occupy ical peptides is known to stabilize helical structures (39), and 2 this effect is enhanced in leucine-zipper and other coiled-coil M. R. Hicks and D. N. Woolfson, unpublished results. 10154 A Designed ␣⫺ to ⫺␤-peptide Switch Downloaded from http://www.jbc.org/ by guest on January 31, 2016 FIG. 5. Properties of the oxidized samples Template-␣T-ox and Template-␣-ox. A, CD spectra for 100 ␮M Template-␣T-ox recorded at 5 °C, 25 mM phosphate (pH 7) before (solid line) and after (broken line) thermal unfolding. B, thermal unfolding curves for 100 ␮M samples of Template-␣T (solid line) and Template-␣T-ox (solid line with open circles). C, transmission electron micrographs of heat-treated and stained Template-␣-ox. a so-called non-hydrogen-bonded site in the hairpin (Fig. 1B). the termini of the peptides using a disulfide bond increased In turn, this would lead to the alignment of complementary, their ability to form amyloid consistent with the possible in- inter-strand hydrophobic interactions (between the a and d volvement of a ␤-hairpin in the fibrillogenesis process. positions of the heptad repeat) and electrostatic pairs (between Conclusions—We have shown that peptides designed and e and g) (Fig. 1B). Furthermore, in this conformation, two of the characterized as a canonical, dimeric leucine-zipper can be Thr residues introduced in Template-␣T should also align induced to switch to ␤-structured states that form amyloid-like across the structure at a favored non-hydrogen-bonded site fibrils. The switches were heat-induced, indeed they occurred (43). With these potential interactions, and if a ␤-structured from the heat-denatured states of the peptides, and they were precursor could seed/promote amyloid-like fibril formation, one facilitated by modifications that raise the ␤-propensity of the might expect that forcing the intra-molecular Cys-Cys bridge sequence. Straight destabilization of the native leucine-zipper would facilitate the ␣ to ␤ transitions in the Template-␣ pep- state did not promote the switch per se; elevated temperatures tides and promote fibrillogenesis. were still required to induce ␤-structure in a mutant that did To probe the role of the intramolecular cross-link, oxidized not form a leucine-zipper dimer at ambient temperatures. This variants for Template-␣, Template-␣T and Template-␣Tu, contrasts with reports for certain globular proteins in which which we distinguish with the suffix “-ox,” were prepared. For amyloid formation correlates with the destabilization of the each peptide, mass spectrometry confirmed the intramolecular native state (18, 20). disulfide bonds, and either sedimentation equilibrium experi- What drives the formation of the ␤-structured states and ments or analytical size-exclusion chromatography was used to amyloid-like fibrils in our designed system? The following are show that the low-temperature states were monomers. At 100 possible contributors. The fact that the transitions in Tem- ␮M and 5 °C Template-␣T-ox was ␣-helical and thermally sta- plate-␣ and Template-␣T were induced by heat is consistent bilized (Fig. 5, A and B). Thermal unfolding converted the with other studies that show the requirement for the polypep- sample to a ␤-structured state (Fig. 5A). Compared with Tem- tides to be partly or fully unfolded to transform to amyloid (9, plate-␣T, this conversion of Template-␣T-ox to ␤-structured 20). However, in our system (as judged by fav-UV CD spectra), aggregates was facilitated; a 100 ␮M sample could be trans- Template-␣Tu was largely unfolded under all conditions, but formed in ⬇20 min at 70 °C. The gels and fibrils from these still required heating to switch state. This requirement for heat samples had all the previously described characteristics con- also suggests the involvement of the hydrophobic effect. This is sistent with amyloid. For Template-␣Tu-ox the disulfide link consistent with our observation that the formation of the did not induce additional structure at low temperature, but the ␤-structured state was accelerated by salt, and foregoing work temperature required to observe the conversion to ␤-structured on other peptides that undergo structural switches (12, 26), in the presence of salt was lowered to 50 °C (compared with and most recently on insulin variants that form fibrils (22). The 70 °C for the reduced peptide). Although we screened a number hydrophobic effect cannot be the only factor at play, however. of conditions, only the oxidized form of Template-␣ showed any This is for several reasons. First, the mutation from Gln to Thr, tendency to form amyloid-like fibrils (Fig. 5C). Thus, tethering which first resulted in the structural switch, is a swap of one A Designed ␣⫺ to ⫺␤-peptide Switch 10155 polar amino acid for another. Second, low pH abrogates the ␣ to comments on this manuscript. D. N. W. is grateful to the BBSRC of the UK and The Wellcome Trust for project and equipment grants. ␤ switch: none of the peptides converted to the ␤-form at pH 2 (data not shown); and the soluble, helical form of Template-␣T REFERENCES could be recovered by re-suspending the fibril-containing gels 1. Skehel, J. J., and Wiley, D. C. (2000) Annu. Rev. Biochem. 69, 531–569 2. Jackson, G. S., and Clarke, A. R. (2000) Curr. Opin. Struct. Biol. 10, 69 –74 in pH 2 buffer. 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T., and DeGrado, W. F. (1990) Science 250, 646 – 651 37. Kim, C. A., and Berg, J. M. (1993) Nature 362, 267–270 ␤⫺structure, specific sequence features can also promote amy- 38. Levine, H. (1993) Protein Sci. 2, 404 – 410 loid-like fibrils. It remains to be seen whether the system will 39. Chakrabartty, A., Doig, A. J., and Baldwin, R. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11332–11336 provide a model for studying amyloid formation in general. 40. Greenfield, N. J., Stafford, W. F., and Hitchcockdegregori, S. E. (1994) Protein Nonetheless, the peptides described should provide a means to Sci. 3, 402– 410 41. Ohnishi, S., Koide, A., and Koide, S. (2000) J. Mol. Biol. 301, 477– 489 unveil systematically features that contribute to ␣ to ␤ struc- 42. Wouters, M. A., and Curmi, P. M. G. (1995) Proteins 22, 119 –131 tural transitions in peptides and proteins. In addition, the 43. Hutchinson, E. G., Sessions, R. B., Thornton, J. M., and Woolfson, D. N. (1998) Protein Sci. 7, 2287–2300 approach that we advocate potentially offers routes to ratio- 44. Kelly, J. W. (2000) Nat. Struct. Biol. 7, 824 – 826 nally designed conformational switches and even new bioma- 45. Kallberg, K., Gustafsson, M., Persson, B., Thyberg, J., and Johansson, J. terials (17, 47, 48). (2001) J. Biol. Chem. 276, 12945–12950 46. Broome, B. M., and Hecht, M. H. (2000) J. Mol. Biol. 296, 961–968 47. Holmes, T. C., de Lacalle, S., Su, X., Liu, G. S., Rich, A., and Zhang, S. G. (2000) Acknowledgments—We thank Chris Kowalczyk and Andrew Smith Proc. Natl. Acad. Sci. U. S. A. 97, 6728 – 6733 for synthesizing the peptides, Drs. Maya Pandya and Julian Thorpe for 48. Lashuel, H. A., LaBrenz, S. R., Woo, L., Serpell, L. C., and Kelly, J. W. (2000) help and advice with the electron microscopy, and Dr. Louise Serpell for J. Am. Chem. Soc. 122, 5262–5277 A Designed System for Assessing How Sequence Affects α to β Conformational Transitions in Proteins Barbara Ciani, E. Gail Hutchinson, Richard B. Sessions and Derek N. Woolfson J. Biol. Chem. 2002, 277:10150-10155. doi: 10.1074/jbc.M107663200 originally published online December 21, 2001 Access the most updated version of this article at doi: 10.1074/jbc.M107663200 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Downloaded from http://www.jbc.org/ by guest on January 31, 2016 This article cites 48 references, 13 of which can be accessed free at http://www.jbc.org/content/277/12/10150.full.html#ref-list-1