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,

A. K. Geim, Rev. Mod. Phys. 81, 109 (2009). 8. D. N. Basov, M. M. Fogler, A. Lanzara, F. Wang, Y. Zhang, Rev.

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: d.n.woolfson@bristol.ac.uk

“...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

http://science.sciencemag.org/

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134 14 JULY 2017 • VOL 357 ISSUE 6347 sciencemag.org SCIENCE

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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

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