BIOCHEMISTRY How do m iniprote ns fold?
Transcript of BIOCHEMISTRY How do m iniprote ns fold?
14 JULY 2017 • VOL 357 ISSUE 6347 133SCIENCE sciencemag.org
By Derek N. Woolfson,1,2,3 Emily G. Baker,1
Gail J. Bartlett1
How does the amino acid sequence of a protein chain determine and main-tain its three-dimensional folded state? Answering this question—a key aspect of the protein-folding problem (1)—would help to explain
how multiple noncovalent interactions con-spire to assemble and stabilize complicated biomolecular structures; to predict protein structure and function from sequence for proteins that cannot be characterized ex-perimentally; and to design new protein structures that do not exist in nature (2). On page 168 of this issue, Rocklin et al. use parallel protein design on a massive scale to create thousands of miniprotein variants and to determine what sequences specify and stabilize these structures (3). The work opens up considerable possibilities for pro-tein folding and design.
Miniproteins are polypeptides shorter than 40 to 50 residues with stable tertiary structures (folds) that contain a limited number of secondary structure elements, such as a helices and b strands. By con-trast, larger proteins have hundreds of amino acids that are often arranged in com-plex three-dimensional structures. Thus, miniproteins simplify the protein-folding problem and potentially allow in-depth ex-aminations of sequence-structure-stability relationships without complications from larger protein contexts. Unfortunately, only a few miniproteins that are stable without covalent cross-links or stabilizing metal ions are currently available for such studies (4).
In their study, Rocklin et al. combine high-throughput DNA synthesis and clon-ing (5, 6) with methods for selecting stably folded proteins (7–9). They implement the latter by expressing libraries of miniproteins on the surface of yeast; tagging the displayed proteins with a fluorescent dye; and discrim-inating between stable and unstable folds through their ability to resist or succumb to protease treatment, respectively (see the
figure). Proteins that survive are rescued by fluorescence-activated cell sorting and then identified by deep sequencing. However, the team’s experiments go beyond a yes/no meas-ure of protein resilience, providing a semi-quantitative measure of stability.
To demonstrate the approach, the authors first apply their method to many variants of a small number of known miniproteins. With the method established, they turn their atten-tion to four classes of de novo miniproteins, which they design computationally using Rosetta (10): aaa, babb, abba, and bbabb folds, where each Greek letter represents an a helix or a b strand in the peptide string.
To cover swaths of sequence space, the team generate diverse libraries with minimal se-quence identity between members.
They then use iterative rounds of protease selection and stability scoring, testing differ-ent hypotheses, and introducing tweaks to the design methods and protocols at each stage. The value of these tweaks is apparent from the improved success rate—the proportion of stable proteins in the starting library—which reaches 87% for one target. However, both the initial and final design success rates depend critically on the fold being targeted, with the aaa fold proving easiest and the abba fold most difficult to optimize.
Through sequence analyses of many thou-sands of these new and also existing mini-protein folds from other studies, the authors highlight several key sequence and structural features. First, a long-established basic tenet of protein folding and design shines through: the importance of burying nonpolar surfaces. This is not surprising, but Rocklin et al. quan-tify the effect, showing that stable variants require more than 30 Å2 for each residue of buried hydrocarbon.
Second, of the initial computational de-signs, those containing peptide fragments ge-ometrically similar to ones known from the
thetic conductors is the absence of a band gap in the former. Electrons can strongly in-teract with holes in gapless graphene (6), and this process changes the “sign” of the velocity renormalization correction compared with the case of electron-electron interaction.
Strong electron-hole interactions may cause the electronic liquid in graphene to become highly viscous (10). The mutual vis-cous friction forces electrons and holes to move together, so that the effective charge contributing to the low-frequency optical response of the electron liquid is dimin-ished. In theory, this effect should inhibit plasmons but enable another type of collec-tive excitation—energy waves or “demons” (11)—to exist at small v and q (see the fig-ure). The thermal photocurrent mapping technique devised by Lundeberg et al. (2, 4) appears to be particularly promising for detection of these elusive modes.
Lundeberg et al. point out that their method of determining the nonlocal complex conductivity s9(v,q) + is99 (v,q) is applicable to other quantum materials, including low-dimensional conductors, superconductors, and Weyl semimetals. A technical precondition for such experi-ments is the ability to use nano-optical imaging at cryogenic temperatures, which recently became available (12). Plasmonic imaging in graphene at liquid helium tem-perature are also highly desirable because scattering by phonons in this regime will be reduced, whereas the observables as-sociated with many-body physics are ex-pected to be enhanced. We anticipate that future studies will address yet another unresolved issue pertaining to the analy-sis of the linewidth of plasmonic modes in graphene that is determined by the ratio s9(v,q) /s99 (v,q). Plasmonic images, includ-ing those reported in (2–4), prompt us to reimagine the sheer scope of unresolved problems that can be tackled with this in-novative experimental approach. j
REFERENCES
1. D. N. Basov, R. D. Averitt, D. van der Marel, M. Dressel, K. Haule, Rev. Mod. Phys. 83, 471 (2011).
2. M. B. Lundeberg et al., Science 357, 187 (2017). 3. D. N. Basov, M. M. Fogler, F. J. Garcia de Abajo, Science 354,
195 (2016). 4. P. Alonso-González et al., Nat. Nano. 12, 31 (2017). 5. D. C. Elias et al., Nat. Phys. 7, 701 (2011). 6. J. González, F. Guinea, M. A. H. Vozmediano, Phys. Rev. Lett.
77, 3589 (1996). 7. A. H. Castro Neto, F. Guinea, N. M. R. Peres, K. S. Novoselov,
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Mod. Phys. 86, 959 (2014). 9. C. F. Hirjibehedin, A. Pinczuk, B. S. Dennis, L. N. Pfeiffer, K.
W. West, Phys. Rev. B 65, 161309 (2002). 10. R. Krishna Kumar et al., arXiv:1703.06672v1 (2017). 11. Z. Sun, D. N. Basov, M. M. Fogler, Phys. Rev. Lett. 117,
076805 (2016). 12. A. S. McLeod et al., Nat. Phys. 13, 80 (2017).
10.1126/science.aan5361
BIOCHEMISTRY
How do miniproteins fold?A high-throughput study yields libraries of miniproteins that help to explain how proteins are stabilized
1School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK. 2School of Biochemistry, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK. 3Bristol BioDesign Institute, University of Bristol, Life Sciences Building, Tyndall Avenue, Bristol BS8 1TQ, UK. Email: [email protected]
“...Rocklin et al. have taken high-throughput, data-driven protein de sign, selection, and optimization to new heights…”
Published by AAAS
on July 20, 2017
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LITDGLA
AAQFV
TDETITLVGNFTGFAGVAEQSGLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVSLGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGVLSSLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFQLAGRPGIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLLVVDATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPDAATGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLLITRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADDAPDAPDAPDAPDAPDAPDAPDAPDAPDAPQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSQIHSRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLTLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLATLAGLALLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLLLVLPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPTG
HGLHGLHGLHGLHGLHGLHGLVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVVVIVALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAALIAGIRNSGAVITAM
FAAQFV
RSSDAG
ALV
GVEQSGLLSVSLAS
RSSGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVAFAGVLSSDAGYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFQLAGIAGIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLSANLPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDPVDATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTEAAHEAAHEAAHIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPIIDPAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYWFIIASTFLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLVTGLITRTLTEPRLAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADAAPQIHSRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLTLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAILLALLVLPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPTGSPTGSPTGSPTGSPTGSPTGSPTGSPTGSPTGSPTGSVFIHGLVVIVALIVSGVV
EQLGNLLPLTAV
LVDAG RPPIAG LV
DRLVASL
RNSGAVITMFFAA
LLPLTAVLDETITAHSLLDLVGNFTGFAPLGV
LGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVALGVAEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGEQSGLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVLLSVSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSLASSSGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALGGALVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVVFTVAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGAFAGVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSVLSSL
VDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGVDAGYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLYVVLIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAIPLAGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFGLVFQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGQLAGRPPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGPIAGIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAIATAFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVFAAVSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSGGFSANLLVDATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAATLAGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTGLSTEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHEAAHIIDPATGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYTGNYWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIWFIIASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFASTFLVTGLVTGLVTGLVTGLVTGLVTGLVTGLRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTRTLTEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLEPRLAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANAHANTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADTVADASIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSIHSRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKRAMKWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLWTGLTLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAITLAILVLVLVLVLVLVLVLVLVLVLPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPNDAPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPLRHPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGPTGLVVIVALIAGRNSGAVIT
MTRTHGWLARLEQLGNRLPHPTLLFVWFCLLLLPLTAVLGALDVTATHPLTDETITAHSLLDADGLRYLFTTLVGNFTGFAPLGVVLVAMLGLGVAEQSGLLSVSLASLVRRSSGGALVFTVAFAGVLSSLT
RPLVDRLVASLLVLAVTM
YLVLMFFAAQFVAWFNY
ATHPLTDETITAHSLLDADGYLFTTLVGNFTGFAPLGVVLMLGLGVAEQSGLLSVSLASLRSSGGALVFTVAFAGVLSSL
VVLIPLAGLVFQLAGRATAFAAVSGGFSANLL
GPVDATLAGLSTEAAHIIDPDRTVAATGNYWFIIASTFLVTGLVTLITRTLTEPRLAHANTVADASVDAPQIHSRAMKWTGLTLAILLAGLALLVLPNDAPLRHPTGSVL
VVIVALIAGICGAQFRNSGAVITAMEVT
YLVLMFFAAQFVAWFN
VDPIGPTVTLVDVDVDAPVDAPVDAPVDVDAPVDAPVDAPVVDAPVDAPVDAPVDAPVDVDAPVVDAPVDAPVDAPVDAPAGAGAGAGAGAGAGAGAAAGAGAGAAGAGGAGLAAGAGLAAGAGLAAGLAAGLAAGLAAAGLAAGAGAGAGAGAGLAAGLAAGLAAGLAAGLAAAGLAAGLAAGAGLAAGLAAGAGLAAGLAAGLAAGLAGAAGLAAGAGLAAGLAAGAGLAAGAGAGLAAAGLAAGAGAGAGLAAGLAAGLAGAGAGLAAGLAAGAGLAAGLAAGLAAGLAAGAGLAAGLAAGLAAGLAAGLAAGLAAGLAGSGSGSGGSGGGSSGSPFGSPFGSPFGSPFGSPFGSPFSGSGSGSPFGSPFGSGSPFGSPFGSGSGSPFGSPFGSPFGSPFGSPFGSPFGSGSPFGSPFGSGGSPFGSPFGSPFGSPFGSPFGSPFGSGSPFGSGSGGSPFGSPFGSGSPFGSGSGSPFGSGSPFSYGGYGYGYGGYGYGRVYGYGYGYGRVYGRVYGYGYGYGRVYGRVYGRVYGYGRVYGRVYGRVYGRVYGYGRVYGYGYGYGRVYGRVYGRVYGYGRVYGYGYGYGRVYGYGRVYGRVYGRVYGRVYGRVYGRVASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAASMAGYGYGYGYGYGYGYGYGYGYGY
VDAGYVPIAGIAGPVDATTVAATGTLITRTVDAPQIAGLAAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLAGLALLGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFGSPFIHYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVYGRVASMAASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMASMAGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGY
IASASRLRLAAA
RAMKRAMKRAMKRAMKVLVLPNDPND
IHGLIHGLIHGLVVVVSGQFSGQFGQFFFFFFFFFFFFFFFFFFFFFIHIHIHVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFSGQFAAAAAAAAGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGY
GTEAA
ST
2 Libraries of amino acid sequences are generated to best ft these structures. Those sequences are synthesized via high-throughput DNA synthesis and cloning.
3 The resulting proteins are expressed on the surface of yeast.
4 Stable variants are selected based on resistance to treatment with protease.
5 The sequences of stable variants are analyzed to determine sequence–stability relationships, which are fed back into the design cycle.
1 Miniprotein structures are designed computationally using a fragment-based approach in Rosetta.
Protease
treatment
Protein Data Bank of protein structures fared
better in the selection process than those
with more geometrically distant matches; i.e.,
the former gave more stable sequences. This
could be a consequence of using Rosetta to
achieve the design frameworks, given that it
is a fragment-based design approach. In the
future, it will be interesting to see how start-
ing points from parametric and other design
approaches perform (11–13).
Third, one relationship not included or
tweaked during the iterative process—it
simply emerges from the analysis—is the
importance of having charged side chains
at the termini of the a helices that oppose
the terminal partial charges of the helices.
This concurs with studies of model peptides
that form freestanding a helices in solution,
where helix formation is attributed to local
capping effects (14).
Despite the impressive and expansive na-
ture of the study, there are gaps to fill and
more steps to take. Although the authors
have characterized many sequences for the
target designs by circular dichroism spec-
troscopy, size-exclusion chromatography,
and thermal and chemical denaturation, and
have verified a small number of structures
by nuclear magnetic resonance spectros-
copy, more high-resolution structural details
would be welcome—for instance, from x-ray
crystallography. Such structures would allow
sequence-structure-stability relationships to
be rationalized in terms of specific noncova-
lent interactions that likely underlie them.
For example, the study points to stabilizing
roles for aromatic residues at surface-exposed
sites of a helices and b strands in minipro-
teins, which hint at noncovalent interactions
particular to this class of side chain.
In an unrelated but pertinent study, Baker
et al. recently designed, characterized, and
interrogated another monomeric minipro-
tein, PPa. This miniprotein has a compact
structure comprising a polyproline II
helix and an a helix that are connected
by an intervening loop (15). A key de-
terminant of PPa’s stability comes
from intimate CH-p interactions be-
tween tyrosine residues of the a helix
and proline residues of the buttress-
ing polyproline II helix. Studying the
role and interplay of these and other
noncovalent interactions will be critical
for feeding back into and improving com-
putational design methods.
In their study, Rocklin et al. have taken
high-throughput, data-driven protein de-
sign, selection, and optimization to new
heights, bringing us closer to solving as-
pects of the protein-folding problem. A
combination of high-throughput studies
of the sequence-structure-stability rela-
tionships described by Rocklin et al. and
drilled-down, fully quantitative examina-
tions of the noncovalent interactions within
(mini)proteins will bring us even closer to
solving this long-standing problem. In turn,
this will facilitate better engineering of nat-
ural and de novo proteins. j
REFERENCES AND NOTES
1. K. A. Dill, J. L. MacCallum, Science338, 1042 (2012). 2. W. R. Taylor, V. Chelliah, S. M. Hollup, J. T. MacDonald,
I. Jonassen, Structure 17, 1244 (2009). 3. G. J. Rocklin et al., Science 357, 168 (2017). 4. S. H. Gellman, D. N. Woolfson, Nat. Struct. Biol.9, 408
(2002). 5. S. Kosuri, G. M. Church, Nat. Methods 11, 499 (2014). 6. M. G. F. Sun, M. H. Seo, S. Nim, C. Corbi-Verge, P. M. Kim,
Sci. Adv.2, e1600692 (2016). 7. P. Kristensen, G. Winter, Fold Des. 3, 321 (1998). 8. V. Sieber, A. Pluckthun, F. X. Schmid, Nat. Biotechnol. 16,
955 (1998). 9. M. D. Finucane, M. Tuna, J. H. Lees, D. N. Woolfson,
Biochemistry 38, 11604 (1999). 10. R. Das, D. Baker, Annu. Rev. Biochem. 77, 363 (2008). 11. P. S. Huang et al., Science 346, 481 (2014). 12. A. R. Thomson et al., Science346, 485 (2014). 13. T. J. Brunette et al., Nature 528, 580 (2015). 14. E. G. Baker et al., Nat. Chem. Biol. 11, 221 (2015). 15. E. G. Baker et al., Nat. Chem. Biol. 13, 764 (2017).
ACKNOWLEDGMENTS
D.N.W. and E.G.B. are supported by a Biotechnology and Biological Sciences Research Council–ERASynBio grant (BB/M005615/1); D.N.W. and G.J.B. are supported by the European Research Council (340764); and D.N.W. holds a Royal Society Wolfson Research Merit Award (WM140008).
10.1126/science.aan6864
Protein design cycle Rocklin et al. use an iterative design cycle to create stable miniproteins. After initially designing miniprotein folds using computational tools, they express them and test their stability, followed by further optimization cycles.
Published by AAAS
on July 20, 2017
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How do miniproteins fold?Derek N. Woolfson, Emily G. Baker and Gail J. Bartlett
DOI: 10.1126/science.aan6864 (6347), 133-134.357Science
ARTICLE TOOLS http://science.sciencemag.org/content/357/6347/133
CONTENTRELATED http://science.sciencemag.org/content/sci/357/6347/168.full
REFERENCES
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