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    Reliable folding of hybrid tetrapeptides into short β-hairpins

    2022-03-14 09:28:16XuYiSunYulongZhongYoHuLiDnilMillrSgrButtnXingXingWuYukunZhngQunTngHongWiTnJinZhuRuiLiuEvZurkZhongLinLuBingGong
    Chinese Chemical Letters 2022年1期

    Xu-Yi Sun,Yulong Zhong,Yo-Hu Li,Dnil P.Millr,Sgr Buttn,Xing-Xing Wu,Yukun Zhng,Qun Tng,Hong-Wi Tn,Jin Zhu,Rui Liu,Ev Zurk,Zhong-Lin Lu,?,Bing Gong,?

    aCollege of Chemistry,Beijing Normal University,Beijing 100875,China

    bDepartment of Chemistry,University at Buffalo,The State University of New York,Buffalo,NY 14260,United States

    cDepartment of Chemistry,Hofstra University,Hempstead,NY 11549,United States

    dAcademy of Chinese Medical Science,Henan University of Chinese Medicine,Zhengzhou 450046,China

    eChengdu Institute of Organic Chemistry,Chinese Academy of Sciences,Chengdu 610041,China

    1These two authors contributed equally to this work.

    ABSTRACT Five hybrid tetrapeptides,each consisting a central dipeptide segment of α-amino acid residues flanked by two aromatic γ-amino acid residues,are found to fold into well-defined β-hairpin conformations as shown by NMR,computational study,and X-ray structures.The turn loop of this β-hairpin motif accommodates different two-residue α-amino acid sequences from the highly flexible Gly-Gly,to the more restricted D-Pro-Gly.The presence of α-amino acid side chains enhances the stabilities of the β-hairpins with the exception of D-Pro-Gly-which results in destabilization.Based on this hairpin/turn motif,a variety of different dipeptide sequences of α-amino acids which rarely occur in β-turns can be introduced and presented as two-residue loops.

    Keywords:β-Hairpin β-Turn γ-Amino acid NMR X-ray

    As a major class of protein secondary structures,reverse turns are sites of chain reversal responsible for the globular character of a protein[1,2].β-Turns,i.e.,tight turns consisting of two amino acid residues,are frequently found in hairpin loops of globular proteins[3-5].The design of type II’D-Pro-Glyβ-turn[6-9],type I’Asn-Gly[10-12],Aib-D-Ala[13]β-turns,and other such as aza-amino acids[14]have facilitated the formation and stabilization of isolatedβ-hairpins consisting ofα-amino acids,and have played crucial roles in initiating the folding of peptides and proteins[15,16].Stableβ-turns also exhibit important biological functions such as serving as epitopes in protein–protein[17-20]and protein–nucleic acid[21-25]interactions.

    Interests in developing foldamers have led to the creation of many folding oligomers containing unnatural building blocks that adopt defined conformations including sheets and turns[26-30].For example,aromatic residues derived from 5-amino-2-methoxybenzoic acid were incorporated intoβ-strands by Nowick to enhance the stability of templated and cyclizedβ-sheets[31].β-Turns based on nipecotic acid developed and those containingβ?2,3-amino acid residues reported by Seebach[32,33]were shown to facilitate the formation ofβ-hairpin structures ofβ-peptides.Besides,β-hairpins containing centrally locatedα/γandα/δ-hybrid dipeptide loops that connect oligopeptide strands ofα-amino acid residues were also reported[34].

    We created oligoamide strands consisting of alternatingmetalinked aromatic units andα-amino acid residues[35].Such oligoamides strands,as mimics ofβ-strands,associate into doublestranded sheets with stabilities proportional to the number of inter-strand H-bonds[36-38].A recent attempt to enhance the diversity of our H-bonded duplexes,based on the incorporation of glycine andβ-alanine,the most flexibleα- andβ-amino acid residues,into the oligoamide strands,led to hybrid peptide 1(Fig.1a)that was found to adopt a stably folded hairpin,i.e.,an expandedβ-turn,containing aβ-Ala-Gly-dipeptide loop flanked by two doubly H-bonded aromaticγ-amino acid residues[39].Subsequent studies revealed that such an expandedβ-turn motif is capable of accommodating a variety of different combinations ofα/β-dipeptide sequences[40,41].

    Fig.1.(a)Hybrid peptide 1 shown as its expanded β-turn conformation.(b)Hybrid peptides 2a-2e shown as their likely β-turn conformations.The amino acid residues are labeled in red.The selected hydrogens are labeled in blue.

    To probe whether theβ-turn motif adopted by 1 and its analogous hybrid peptides could be extended to constructing tworesidue loops consisting ofα-amino acid residues,four-residue hybrid peptides 2,which differ in theα-amino acid sequences of their central dipeptide segments,are designed(Fig.1b).Each of peptides 2,if folding into the conformation as shown,will give aβ-hairpin consisting of,from the N- to the C-termini,four consecutive residues with positionibeing an aromaticγ-amino acid,positionsi+ 1 andi+ 2 being twoα-amino acid,andi+ 3 being another aromaticγ-amino acid,residues.By studying the folding of 2,we hope to address the following questions:Could such aβturn motif accommodate differentα-amino acid residues? How do differentα-amino acid residues influence the stability of the correspondingβ-turns?

    Herein we report that the folding of peptides 2a-2e,with residuesi+ 1 andi+ 2 being Gly-Gly,L-Ala-Gly,L-Phe-Gly,L-Ala-LAla and D-Pro-Gly,follows a general pattern by adoptingβ-hairpin conformations as shown by results from 1D and 2D1H NMR studies and confirmed by the crystal structures of 2a-2d.The stabilities of the hairpin conformations are found to vary with theα-amino acid sequences of the dipeptide loops.

    The1H NMR spectra of 2a-2e from 0.1 mmol/L to 25 mmol/L in CDCl3reveal sharp peaks,except for protonaof 2e(Fig.S1 in Supporting information).The well dispersed1H resonances of these hybrid peptides indicate that,although having multiple Hbond donors and acceptors,peptides 2a-2e do not engage in noticeable self-aggregation and most likely exist as discrete species adopting defined conformations in solution.Consistent with the adoption ofβ-turn conformations by 2a-2e,amide protonsa,b,dandeshow insignificant changes in their chemical shifts,indicating their involvement in intramolecular H-bonding;in contrast,protonscof all five peptides show much larger downfield shifts,suggesting their engagement in intermolecular H-bonding.

    Comparing the chemical shifts of amide protonsaanddof 2a-2d(Table 1)at 5 mmol/L provides for a qualitative assessment on the relative stabilities of the corresponding H-bonds and thus those of theβ-turns.Compared to that of 2a,the signals of protondof 2b and 2c move downfield,which imply that the H-bonded loops of 2b and 2c are stabilized due to the presence of the L-Ala-and LPhe-side chains;the signal of protondof 2d moves slightly upfield,suggesting that the two L-Ala-residues more or less twist the Hbonded loop and weakens the H-bond involving protond.

    Table 1 Chemical shifts of protons a,d,f,and f1 in CDCl3.a

    The chemical shifts of protonsawhich are away from the Hbonded loops indicate the strength of the H-bonds and also indi-cate the stability of theβ-turns.Relative to that of 2a,the signals of protonsaof 2b-2d move downfield.The resonance of 2d shows the largest downfield shift,followed by that of 2b,and then by 2c,based on a stability order of 2d>2b>2c>2a results.Thus,the presence ofα-amino acid sidechains in hybrid peptides 2b-2d seems to enhance the stability of the hairpins,most likely by imposing stereochemical constraints that limit the conformational flexibility of the turn loops.

    Fig.2.NOEs shown as double-headed arrows,between remote(non-adjacent)protons revealed by the NOESY spectra of(a)2a and(b)2e in CDCl3(5 mmol/L,298 K,400 MHz,mixing time 0.3 s).

    Compared to the sharp resonances of protonsaof 2a-2d,protonaof 2e,which contains a D-Pro-Gly-loop that strongly promotes turn and hairpin conformation in peptides ofα-amino acids[6],gives a broadened peak(Fig.S1e)that moves upfield to 9.551 ppm(Table 1).Besides,the peaks of protonsfandf1of 2e also move upfield to 7.915 and 7.403 ppm,respectively,relative to those of protonsfandf1of 2a-2d(Table 1).Thus,the H-bond involving protonaof 2e seems to be weakened,resulting in a conformation of 2e in which the two aromatic residues and theN- andC-termini undergo dynamic relative motion.

    Peptides 2a-2e were then examined with 2D NMR(NOESY)in CDCl3(Fig.S2 in Supporting information).The NOEs observed with 2a(Fig.2a)are those between protonsaandl,aandf1,dandf,andfandf1,indicating that the two aromatic rings are brought into close proximity by H-bonding involving amide protonsaandd.Besides,the NOE between protonslandmdemonstrates that the two termini of 2a are in close proximity,which is consistent with the expected hairpin conformation.NOEs involving the protons of the Gly-Gly-segment of 2a include those between protonscandb,candd,anddandj,which indicate the presence of a well-defined loop.The NOESY spectra of 2b-2d(Figs.S2b-d)reveal that NOEs share the same overall pattern observed in that of 2a,suggesting that 2b-2d also adopt well-definedβ-turn conformations in CDCl3.

    The NOEs revealed for 2e(Fig.2b)include those between protonscandd,dandj,dand f,fandf1,andlandm,indicating that 2e also adopts aβ-turn conformation.That no NOE between protonsaandf1,oraandlis observed,along with the significant upfield shift of this proton resonance relative to the signals of protonsaof 2a-2d,is consistent with the weakening of the H-bond involving protonaof 2e.

    Fig.3.Crystal structures of(a)2a,(b)2b,(c)2c,and(d)2d Two independent molecules with their main chain atoms adopting enantiomeric turn types having are found for 2a,2c,or 2d For clarity,all hydrogen atoms except for those of the amide groups,are removed.

    Single crystals of 2a-2d were obtained,which led to the determination of the X-ray structures of these hybrid peptides.As shown in Fig.3,each of the four hybrid peptides,regardless of its two-residue sequence involving residuesi+ 1 andi+ 2,folds into a typicalβ-hairpin conformation that is held together by two intramolecular H-bonds involving amide protonsaanddof residuesiandi+ 3.Although the two-residue sequences Gly-Gly,L-Ala-Gly,L-Phe-Gly and L-Ala-L-Ala-do not have highβ-turn propensities[4,5],peptides 2a-2d nevertheless fold intoβ-hairpins,which demonstrate the general applicability of thisβ-turn motif for accommodating different loop sequences.

    Based on the crystal structures of 2a-2d,dihedral anglesφandψof residuesi+ 1 andi+ 2 in the turns are obtained,which reveal theβ-turn types(Fig.3).The crystal structure of achiral 2a(CCDC:2,053,064)reveals two independent molecules adopting the enantiomeric type I and type I’β-turn conformations(Fig.3a).With its chiral L-Ala-residue,peptide 2b(CCDC:2,053,065)exists as two independent molecules with very similarφandψangles and folds into a type IIβ-turn(Fig.3b).Interestingly,peptides 2c(CCDC:2053066)(Fig.3c)and 2d(CCDC:2053067)(Fig.3d),although having one(L-Phe)and two(L-Ala)chiral residues,respectively,still fold into both types I and I’β-turns,i.e.,the presence of chiralα-amino acid residues is incapable of biasing the main chain atoms of 2c or 2d to settle on one of the two enantiomeric turn types.

    Fig.4.Energy-minimized structures of the hairpin conformations of(a)2e and(b)2b.The isopentyloxy side chains are replaced with methoxy groups.For clarity,all hydrogen atoms except for those of the amide groups,are removed.

    Fig.5.Changes in chemical shifts(Δδ)amide protons a and d(400 MHz)of 2a-d(5 mmol/L)(a)from 0—45 °C in CDCl3(Δδ=δ(45 °C)–δ(0 °C))and(b)in mixed solvents containing 0 to 20% DMSO–d6 in CDCl3 at 25 °C(Δδ=δ(20% DMSO)–δ(0% DMSO)).

    Attempts to grow single crystals of peptide 2e have been unsuccessful.The conformation of 2e was computationally probed using the ADF and VASP software packages with revPBE-D3[42-44].The most stable conformation of 2e is aβ-hairpin in which the planes of the two aromatic residues have a sharp dihedral angle of~60°(Fig.4a).Besides,the benzene ring of residueiand the amide group linking residuesiandi+ 1 deviate from coplanarity,with a dihedral angle of~50°.In contrast,the optimized conformation of 2b shows a much less curved structure,with a dihedral angle of~117° between the benzene rings of the two aromatic residues(Fig.4b).The benzene ring of residueiand the amide group between residuesiandi+ 1 are coplanar.The twisted conformation of 2e seems to be caused by the need to avoid steric hindrance between the five-membered ring of the Pro-residue and the adjacent methoxy sidechain of aromatic residuei.As a result,the two H-bonds involving protonsaanddare slightly longer,and thus weaker,in 2e than those in 2b.

    Quantum molecular dynamics calculations on 2b and 2e in aNVTensemble(298 K,Supporting information for movies)revealed that the length of the H-bond involving protonaof 2b fluctuated in a narrow range(~1.7–2.5 ?A),while the H-bond of 2e involving protonawas drastically more dynamic and fluctuated over a much larger range(~1.7–3.8 ?A)(Fig.S7 in Supporting information).Thus,structural deformation caused by the D-Pro-residue results in weakened H-bonding interactions and thus lowered stability for the hairpin of 2e.

    The stabilities of the folded structures of 2a-2e were further examined by comparing the effects of temperature and solvent polarity on the strength of H-bonds involving protonsaandd(Figs.S3 and S4,Table S1 in Supporting information).As shown in Fig.5a,from 0 to 45 °C,the signals of protonsaanddall move upfield.The signal of protonaof 2e shows an upfield shift(0.43 ppm)that is significantly larger than those of the other four peptides,and those of protonsaof 2a-2d shift within a much narrower range of 0.06 to 0.14 ppm.Among protonsd,that of 2e shows the largest upfield shift(0.21 ppm),while those of 2a-2d are much smaller,ranging from 0.05 to 0.10 ppm.The temperature-dependent upfield shifts of amide protonsaanddindicate that,the presence of the D-Pro-residue in 2e weakens the H-bonds involving protonsaanddmuch more significantly than in 2a-2d.Among 2a-2d,the presence of side chains in theβ-turn loops of 2b-2d slightly weakens the H-bonds involving protonsa,while enhancing the H-bonds involving protonsd.

    Fig.6.(a)Tetrapeptides 3a-c differing in their central residues(highlighted in reddashed box)flanked by the same L-Val-and L-Leu-residues.(b)NOEs(red doubleheaded arrows)between remote protons revealed by the NOESY spectrum of 3c(5 mmol/L in CDCl3,400 MHz,mixing time:0.3 s).

    In mixed solvents containing 0 to 20% DMSO–d6in CDCl3(Fig.5b),the signals of both protonsaanddof 2a-2e show downfield shifts.The shifts of protonsaanddof 2e,being 0.12 and 0.19 ppm,respectively,are much larger than those of the other four peptides,confirming that the protonsaanddare more exposed in 2e,i.e.,the corresponding H-bonds are less shielded,leading to a confirmation of 2e that is less stable than those of 2a-2d.The downfield shifts of protonsaanddof 2a are larger than those of protonsaof 2b-2d,indicating that theα-amino acid side chains of 2b-2d enhance the stabilities of these threeβ-turns,a conclusion that is consistent with the one based on comparing the chemical shift values of protonsa.

    The critical role played by the aromaticγ-amino acid residues in promoting the folding of peptides 2 was demonstrated by comparing 3a-3c which differ in their central dipeptide sequences but share the same L-Val-and L-Leu-terminal residues(Fig.6a).Peptide 3c is known to fold into aβ-hairpin conformation that is promoted by its D-Pro-Gly-loop sequence[6].As shown in Fig.S5 and Table S2(Supporting information),from 0.1 mmol/L to 25 mmol/L in CDCl3,the signals of amide protonsb,c,anddof 3a show noticeable downfield shifts of~0.56 to 0.77 ppm and that of protonashows a very small shift(~0.02 ppm);while all four amide protons of 3b undergo very large downfield shifts from~1.61 to 1.74 ppm.Consistent with the folding of 3c into aβ-hairpin,the downfield shifts of amide protonsaanddof 3c,being 0.13 and 0.05 ppm,are much smaller than those of 3a and 3b(except for protonaof 3a),which also suggests that 3a and 3b undergo intermolecular Hbonding interaction and do not fold like 3c.

    The NOESY spectrum of 3c(Fig.S6 in Supporting information)reveals NOEs between remote protonsaandd,dandi,andeandf.In contrast,no NOEs between the corresponding remote protons,especially between protonsaandd,andeandf,are found in the NOESY spectra of 3a and 3b,showing that 3a and 3b,unlike 3c,do not fold intoβ-hairpin conformations.

    In summary,hybrid tetrapeptides 2a-2e,each has a central dipeptide segment ofα-amino acid residues and two aromaticγamino acid residues,fold into well-definedβ-turn hairpins.Results from 1D and 2D NMR,and computational studies,along with crystal structures,demonstrate that the five different two-residue sequences ofα-amino acid residues are all incorporated as the turn loops of theβ-hairpins.D-Pro-Gly,a turn sequence that strongly promotes the adoption ofβ-hairpin conformations by peptides ofα-amino acids,results in aβ-turn with the lowest stability.Compared to peptide 2a which has a Gly-Gly-loop,peptides 2b-2d with L-Ala-Gly,L-Phe-Gly and L-Ala-L-Ala-loops,showed enhanced stabilities.The inability of peptides 3a and 3b,which shares their central dipeptide segments with 2a and 2d,respectively,demonstrates the critical importance of the two aromaticγ-amino acid residues in ensuring the adoption of hairpin conformations by peptides 2a-2e.The same folding pattern of the five hybrid peptides has offered a reliable hairpin motif capable of accommodating different two-residue sequences ofα-amino acids,which will greatly enhance the diversity ofβ-turns andβ-hairpins.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China(No.21778012 to Z.L.Lu,21801020 to R.Liu),the American Chemical Society–Petroleum Research Fund(PRF#58364-ND7,to B.Gong),and the Center for Computational Research(CCR)(to D.P.Miller and E.Zurek),and Hofstra University(to D.P.Miller).

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2021.06.019.

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