Dispersion from Cα or NH : 4D experiments for backbone resonance assignment of intrinsically disordered proteins

Tossavainen, Helena; Salovaara, Santeri; Hellman, Maarit; Ihalin, Riikka; Permi, Perttu

Dispersion from Cα or NH : 4D experiments for backbone resonance assignment of intrinsically disordered proteins

Tossavainen, Helena,Salovaara, Santeri,Hellman, Maarit,Ihalin, Riikka,Permi, Perttu

Published: 2020
Publisher: Springer
Repository: jyx.jyu.fi
Language: en

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This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ Dispe sion om Cα o NH : 4D expe imen s o backbone esonance assignmen o in insically diso de ed p o eins © The Au ho (s) 2020 Published e sion Tossa ainen, Helena; Salo aa a, San e i; Hellman, Maa i ; Ihalin, Riikka; Pe mi, Pe u Tossa ainen, H., Salo aa a, S., Hellman, M., Ihalin, R., & Pe mi, P. (2020). Dispe sion om Cα o NH : 4D expe imen s o backbone esonance assignmen o in insically diso de ed p o eins. Jou nal o Biomolecula NMR, 74(2-3), 147-159. h ps://doi.o g/10.1007/s10858-020-00299-w 2020 Vol.:(0123456789) 1 3 Jou nal o Biomolecula NMR (2020) 74:147–159 h ps://doi.o g/10.1007/s10858-020-00299-w ARTICLE Dispe sion om Cα o  NH: 4D expe imen s o backbone esonance assignmen o in insically diso de ed p o eins HelenaTossa ainen1 · San e iSalo aa a1· Maa i Hellman1· RiikkaIhalin2 · Pe uPe mi1,3 Recei ed: 30 Augus 2019 / Accep ed: 2 Janua y 2020 / Published online: 13 Janua y 2020 © The Au ho (s) 2020 Abs ac Resonance assignmen o in insically diso de ed p o eins is ema kably challenging due o scan chemical shi dispe sion a ising om con o ma ional he e ogenei y. The challenge is e en g ea e i epea ing segmen s a e p esen in he amino acid sequence. To o wa d unambiguous esonance assignmen o in insically diso de ed p o eins, we p esen iHACANCO, HACACON and (HACA)CONCAHA, h ee Hα-de ec ed 4D expe imen s wi h Cα as an addi ional dimension. In addi ion, we p esen (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D Hα-s a , HN-de ec expe imen s which ha e wo NH dimensions o enhance peak dispe sion in a sequen ial walk h ough C′, NH and HN, and p o ide mo e accu a e NH/HN chemical shi s han hose ha can be ob ained om a c owded 1H, 15N-HSQC spec um. Applica ion o hese 4D expe i- men s is demons a ed using BilRI (165 aa), an ou e -memb ane in insically diso de ed p o ein om he oppo unis ic o al pa hogen Agg ega ibac e ac inomyce emcomi ans. BilRI amino acid sequence encompasses h ee e y simila epea s wi h a 13- esidue iden ical s e ch in wo o hem. Keywo ds Agg ega ibac e ac inomyce emcomi ans· BilRI· Resonance assignmen · In insically diso de ed p o ein· IDP In oduc ion Agg ega ibac e ac inomyce emcomi ans is a G am-nega i e oppo unis ic o al pa hogen ha is linked o pe iodon i is, in ec ion o issues suppo ing he ee h ( o e iews, see Fine e al. 2006; Åbe g e al. 2015; Fine e al. 2019). Al hough A. ac inomyce emcomi ans esides in subgingi al mul ispe- cies bio ilms, i is also able o mig a e o unde lying essels and cause sys emic diseases such as ca dio ascula dis- eases (Koza o e al. 2005; Hy ä inen e al. 2012). The hos esponse o bio ilms is media ed by in lamma o y cy okines. In heal hy junc ional epi helium o oo h he balance o a i- ous cy okines and chemokines ensu es ha he hos de ence wo ks app op ia ely. Some pe iodon al pa hogens a e able o impai he balance. Po phy omonas gingi alis can supp ess he exp ession o chemokine in e leukin(IL)-8 (Takeuchi e al. 2013) and A. ac inomyce emcomi ans bio ilm is able o seques e and in e nalize IL-1β, IL-8 and IL-6, which leads o changes in bio ilm composi ion and me abolic ac i - i y (Paino e al. 2011, 2012; Ahls and e al. 2017). I has been sugges ed ha A. ac inomyce emcomi ans bac e ial IL ecep o I, BilRI is associa ed wi h his seques e ing ac i i y (Paino e al. 2013; Ahls and e al. 2017). BilRI is an ou e memb ane lipop o ein able o bind IL-1β, IL-8 and IL-10 and umo nec osis ac o (TNF)-α (Ahls and e al. 2017). Due o i s a he low binding a ini y i is assumed ha BilRI ac s by concen a ing cy okines on cell memb anes, which a e hen ans e ed o o he componen s o he up ake sys em (Paino e al. 2013; Ahls and e al. 2017). We ha e engaged in he s uc u al cha ac e iza ion o BilRI. BilRI is an in insically diso de ed p o ein (IDP), as dem- ons a ed by i s 1H, 15N HSQC spec um, which displays e y limi ed signal dispe sion (Ahls and e al. 2017). In he HN dimension hei dispe sion is only 0.63ppm. This a ises om a ea u e ypical o IDPs, namely a biased amino acid Elec onic supplemen a y ma e ial The online e sion o his a icle (h ps ://doi.o g/10.1007/s1085 8-020-00299 -w) con ains supplemen a y ma e ial, which is a ailable o au ho ized use s. * Pe u Pe mi pe u.pe [email p o ec ed] 1 Depa men o Chemis y, Nanoscience Cen e , Uni e si y o Jy äskylä, Jy äskylä, Finland 2 Depa men o Biochemis y, Uni e si y o Tu ku, Tu ku, Finland 3 Depa men o Biological andEn i onmen al Science, Uni e si y o Jy äskylä, Jy äskylä, Finland 148 Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 composi ion wi h a p onounced numbe o pola o cha ged amino acids, low numbe o bulky hyd ophobic amino acids and lack o a oma ic amino acids (Dyson 2016). BilRI amino acid sequence is domina ed by alanine (23%), lysine (14%) and aspa ic acid (13%) esidues. Howe e , unlike he gen- e al end (Dunke e al. 2001), BilRI sequence con ains only one p oline and one glycine. BilRI is posi ioned among IDPs in he mean ne cha ge-hyd opa hy plo (U e sky e al. 2000) (Fig.1a). The pauci y in he a iabili y o esidue’s nea es neighbo s na ows down he chemical shi ange o a pa - icula amino acid ype, o example he e a e 12 Lys-Asp- Ala iple s in he sequence, which mos likely esul s in close chemical shi s o he middle aspa ic acid. Addi ion- ally, he BilRI sequence con ains h ee e y simila segmen s o abou o y esidues. The longes iden ical s e ch is o 13 esidues, p esen in wo o hese epea s, co e ing esidues 71–82 and 111–122 (Fig.1b). The 1H, 15N HSQC spec a o IDPs ypically being e y challenging o sc u inize, he esonance assignmen mos o en elies on spec a o he han he classical HN-de ec ed expe imen s widely used o olded p o eins ( o e iews see e.g. Sa le e al. 1999; Pe mi and Annila 2004). Indeed, he con en ional app oach based on he HNCACB and HN(CO)CACB/CBCA(CO)NH expe imen s ha link he in a- and sequen ial 13Cα and 13Cβ chemical shi s o 1HN and 15N equencies, a e e y ine icien o many IDPs due o se e e clus e ing o alipha ic ca bon chemi- cal shi s o each esidue ype. Ins ead, co ela ion o 15N and 13C′ equencies p o ide much be e esul s o IDPs (Yao e al. 1997; Män ylah i e al. 2009; Be mel e al. 2012) Ano he obs acle a ises om he inc easing chemical exchange a e o amide p o ons wi h wa e a alkali pH and/o ele a ed measu emen empe a u e (Män- ylah i e al. 2010). Ye ano he challenge o he assign- men o igina es om he abundancy o p oline esidues in IDPs. As an N-subs i u ed esidue, p oline lacks he amide p o on, which esul s in gaps du ing he esonance assignmen p ocedu e. Al hough his can be a bene i in he case o globula p o eins, i signi ican ly hampe s he esonance assignmen o diso de ed sys ems (Hellman e al. 2014). Se e al di e en app oaches ha e been p o- posed o o e come hese obs acles imposed du ing he assignmen p ocedu e. These include inc ease o dimen- sionali y om con en ional 3D o 4–7D spec a (Fio i o e al. 2006; Mo áčko á e al. 2010; No áček e al. 2011; Kazimie czuk e al. 2013; B u sche e al. 2015) as well as de ec ion o non-exchangeable spins 13C′ and 1Hα ins ead o 1HN (Be mel e al. 2006a, 2009; 2012; Män ylah i e al. 2010, 2011; Pe mi and Hellman 2012). Ou g oup has been eso ing bo h o Hα-s a , HN-de ec pulse schemes (Män ylah i e al. 2009; Hellman e al. 2014) o comple e Hα-de ec ion expe imen s (Män ylah i e al. 2010, 2011; Pe mi and Hellman 2012) o he assignmen o IDPs, which o e come hu dles associa ed wi h p oline assign- men and exchange b oadening a alkali pH o ele a ed empe a u e. In addi ion o educed suscep ibili y owa ds sol en exchange induced lineb oadening, he Hα chemical shi is ex emely aluable in s uc u al analysis. The Cα, Hα and C′ shi s a e pa icula ly sensi i e o he ϕ/ψ angles o he p o ein backbone and hus he mos in o ma i e in he es ima ion o seconda y s uc u e con en in an IDP (Bo che ds and Daughd ill 2018). Comp ehensi e assignmen s allow o de ailed, esidue- speci ic analysis o s uc u e and dynamics (Kon a 2014). The e a e he e o e g ounds o an ex a e o owa ds a mo e comp ehensi e backbone esonance assign- men . IDPs a e o en comp ised o epe i i e amino acid sequences and hence highe dimensionali y in combina ion wi h high esolu ion o e supe io esul s. Howe e , he inc eased dispe sion o signals should no be ob ained, i possible, a expense o sensi i i y. He e we p esen he esonance assignmen o BilRI, whose demanding amino acid sequence necessi a ed de elopmen o a sui e o 4D pulse sequences ha o e supe io signal dispe sion wi h espec o hei well-es ablished 3D coun e pa s wi hou indi ec sampling associa ed sensi i i y loss. b 0.0 0.1 0.2 0.3 0.4 0.5 0.20.3 0.40.5 0.6 BilRI Diso de ed P o eins O de ed P o eins Mean Scaled Hyd opa hy Absolu e Mean Ne Cha ge a DDSKTSPQAEQAKTSVSEAK DAVVNAANDV KDATVEAAKD AQNMAADKMV EVKDAISEKM DAMTTQASEM KDAAVEAAKD AKDAAADKMA EVKDAISEKM DAMATQVNEM KDTAAEAVKD AKDAAADKMT EVKDAVSEKMGATATQTNEM KDAVKSETES K GSHM 100 150 160 170140 180 30 40 50 70 80 9060 110 120 130 Fig. 1 a Mean ne cha ge-hyd opa hy plo o BilRI. Da a o he plo we e c ea ed wi h he PONDR p edic o (h ps ://www.pond .com/). b Amino acid sequence o A. ac inomyce emcomi ans BilRI. Amino acids a e classi ied by ype: yellow, small hyd ophilic (A, G, N, P, Q, S, T); g een, hyd ophobic (I, M, V); ed, nega i ely cha ged (D, E) and blue, posi i ely cha ged (K) amino acids. BilRI sequence does no con ain C, F, H, L, R, W o Y. The longes 13- esidue epe i i e segmen s a e unde lined in black, and he b oken unde line indica es a e y simila segmen . The second longes 11- esidue epe i i e seg- men s a e ande lined in blue, and he iple K-D-A, which ecu s welwe imes in he sequence is unde lined in ed. The i s ou esi- dues a e a cloning a e ac 149Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 Ma e ials andme hods P o ein exp ession andpu i ica ion The gene encoding BilRI ( esidues 21–181) was cloned o pET15b ec o (No agen) in o he NdeI and XhoI si es. This leads o soluble ecombinan BilRI p o ein wi h N- e minal His-Tag wi h a h ombin clea age si e. P oduc ion o 13C, 15N labeled BilRI was ca ied ou by ans o ming plasmids in o he BL21(DE3) cells. Cells we e g own in M9 minimal media, supplemen ed wi h 1g/l o 15NH4Cl and 2g/l 13C-d-glucose as he sole ni ogen o ni ogen and ca bon sou ce, espec i ely. Cell cul u e was incuba ed a 37°C and empe a u e was dec eased o 16°C when OD o he cell cul u e eached 0.4 and p o ein p o- duc ion was induced wi h 1mM IPTG when OD o he cell cul u e eached 0.6. Cells we e u he incuba ed a 16°C o Table 1 Da a acquisi ion pa ame e s Expe imen Poin s in F1 (ms) Poin s in F2 (ms) Poin s in F3 (ms) Poin s in F4 (ms) Sampling % Numbe o scans 4D iHACANCO 80 (16.6) 13C′84 (19.1) 15N 48 (3.9) 13Cα1024 (80.1) 1Hα10 4 4D HACACON 64 (13.2) 15N 64 (14.5) 13C′48 (6.9) 13Cα1024 (80.1) 1Hα10 4 4D (HACA)CONCAHA 64 (13.2) 13C′64 (14.5) 15N 48 (6.9) 13Cα1024 (80.1) 1Hα20 4 4D (HACA)CON(CA)NH 74 (26.3) 13C′80 (24.7) 15N 82 (25.3) 15N 1024 (71.2) 1HN7 4 4D (HACA)N(CA)CONH 74 (26.3) 13C′80 (24.7) 15N 82 (25.3) 15N 1024 (71.2) 1HN7 4 3D iHA(CA)NCO 116 (24.0) 13C′200 (45.5) 15N – 1024 (80.1) 1Hα25 8 3D HA(CA)CON 220 (50.0) 15N 230 (46.8) 13C′– 1024 (80.1) 1Hα25 8 3D (HACA)CON(CA)HA 156 (32.3) 13C′220 (50.0) 15N – 1024 (80.1) 1Hα25 8 15 N (ppm) 1 H (ppm) 13 C (ppm) 181K 164A134A 175K 87A39A 53A 71E 57A 104A 137A 144A,, 56E 82A 173A 61A 42A 96E 151E, 162A 179E 38E 45N 55V 106A 146A, 89E 24K 47A 94A 131K 152V 171K,,,,, 105A 112V 145A,, 128N 132D 129E 79K 119K 159K,, 36V 127V 70V 69M 90M, 169E 108K 160M 148K 168N 149M 68K 41D 49D 60D 64M,,, 99K 50V 59K 102K 28Q 111E 30E 43V 174V 136E 138V 95V 37S 156V 67D 83M 62Q 35S 123M 180S 176S 48N 85T 23S 167T 150T 88S 25T 178T 54T 165T 163T 125T 161G 177E 34T 84T 133T,, 107D 147D, 63N 157S, 26S 77S 117S,, 76I 116I, 142K 109M 33K 40K 100D 172D,, , 103D 143D, 21D 80M 92D 130M,,,20M 81D 120M 121D 140D,, ,, 22D 52D 74D 114D 154D,,,, 158E 78E 86Q 118E 166Q,, , 98A 135A, 58A 66A, 72V 91K 126Q,, 44V 75A 115A 122A 155A,, ,, 65A 93A 97A 141A,, 29A 32A 46A 139K,,, 51K 73K 153K, 31Q 170M, 113K 101A 110A 124A,, x x 120 8.28.3 7.0 7.5 8.08.5 a 121 181K 164A 134A 175K 87A 39A 139K 71E 57A 97A 104A 124A 122A56E 75A 82A 173A 61A 42A 96E 162A 179E 38E 66A 166Q 45N 94A 55V 106A 89E 24K 105A 128N 132D 129E 79K 120M 36V 114D 127V 70V 169E 108K 143D 160M, 148K 168N 149M 100D 68K 41D 99K 50V 59K 102K 28Q 111E 30E 43V 116I 174V 136E 138V 95V 77S 37S 156V 157S 147D 67D 83M 62Q 35S 123M 180S 176S 48N 85T 23S 167T 150T 88S 133T 25T 178T, 54T 165T 163T 125T 161G 177E 34T 84T 107D 63N 117S 26S 76I 142K 60D 49D 109M 64M 40K 172D 33K 103D 119K 159K 21D 80M 121D 74D 154D, 81D 20M 22D 158E 86Q 126Q, 78E 118E 146A 98A 135A, 58A 171K 47A 131K 91K 151E 44V 115A 155A 141A 110A 137A 144A 65A 93A 29A 53A, 32A 46A 51K 153K 73K 152V 145A 31Q 113K 112V 101A 1:90M 170M, 18S 172174176178180 ** * 19H * * 123 45 2:69M 3: ,92D 130M 4:52D 5:140D 176.0 123 122 72V 176.2 b 110 115 120 125 8.4 110 115 120 125 Fig. 2 a 2D 1H, 15N-HSQC spec um o BilRI. The peaks a e labeled wi h esidue numbe s and one-le e amino acid codes. C osses indi- ca e peaks ound a lowe con ou le els. Residue numbe ing co e- sponds o ha o whole BilRI p o ein (1–181) al hough he cons uc used was sho e (21–181). b 2D CON spec um o BilRI. The peaks a e labeled wi h esidue numbe and amino acid code o he amide ni ogen in he C′-NH pai . The peak o he only p oline o he BilRI amino acid sequence, which esona es a 172.8 (26Se C′), 138.1 (27P o NH) ppm is no shown. As e isks indica e impu i ies. Bo h 2D spec a we e acqui ed a 800MHz 1H equency, 25°C om a 1mM BilRI sample a pH 6.5 150 Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 16h and collec ed by cen i uga ion. Cells we e dis up ed wi h sonica ion and esul ing supe na an was cla i ied by cen i uga ion wi h 30,000×g. Cla i ied supe na an o BilRI was applied o he 1-mL His G a iT ap column (GE Heal hca e) and he His-Tag was emo ed by h ombin p o ease (GE Heal hca e) diges- ion acco ding o he manu ac u e ’s ins uc ions. P o ease diges ed mix u e was applied o His G a iT ap column. BilRI, wi hou His-Tag, elu ed wi h low- h ough. Flow- h ough was concen a ed o olume o 1ml wi h Vi aspin 2 concen a o . Concen a ed BilRI sample was applied in o he Supe dex 75 16/60 gel il a ion column (GE Heal h- ca e). Bu e used in gel il a ion con ained 20mM sodium phospha e (pH 6.5) and 50mM NaCl (NMR bu e ). F ac- ions wi h pu e BilRI we e pooled and concen a ed o NMR s udies. The gel il a ion was pe o med by using he ÄKTA Pu i ie FLPC pu i ica ion sys em (GE Heal hca e). abc d e 151Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 NMR spec oscopy BilRI NMR expe imen s we e acqui ed using 0.5–1.0mM 15N, 13C labeled p o ein samples in 5/95% D2O/H2O a pH 6.5. Chemical shi s we e e e enced o ex e nal 2,2,-dime- hyl-2-silapen ane-5-sul onic acid (DSS). All da a we e acqui ed a 25°C on a B uke AVANCE III HD 800MHz spec ome e , equipped wi h a TCI 1H/13C/15N c yop obe. In addi ion o he new 4D expe imen s desc ibed he e, he ollowing expe imen s we e used in he esonance assign- men : 2D 1H, 15N-HSQC, cons an ime 1H, 13C-HSQC, 13C-de ec ed 2D CON (Be mel e al. 2006b), 3D HN-de ec ed HNCACB, CBCA(CO)NH, HNCO ( e iewed in (Sa le e al. 1999; Pe mi and Annila 2004) and i(HACA)CO(CA) NH (Män ylah i e al. 2009), 3D Hα-de ec ed HA(CA)CON, iHA(CA)NCO and (HACA)CON(CA)HA (Män ylah i e al. 2010, 2011). All 3D/4D expe imen s we e collec ed using non-uni o m sampling (Table1). Sampling densi ies we e 25% o he 3D expe imen s and 7–20% o he 4D expe i- men s. NMR da a we e p ocessed wi h TopSpin 3.5 (B uke Inc) and analyzed wi h CcpNm Analysis . 2.4.2 (V anken e al. 2005). BilRI chemical shi s ha e been deposi ed o he BMRB da abase (www.bm b.wisc.edu) wi h accession code 27824. Resul s anddiscussion Peaks in he BilRI 2D 1H, 15N-HSQC spec um display o e - whelming o e lap (Fig.2a) and a e y na ow dis ibu ion in he HN dimension. As i was la e e ealed, all bu h ee o he 21 aspa ic acid esidues’ peaks a e loca ed in he middle egion pile o peaks, 8.27–8.38, 121.2–121.9ppm, oge he wi h 11 o he 13 me hionine esidues’ amide peaks. The Asp and Me α signals hea ily o e lap also in he 1H, 13C CT-HSQC (Suppl. Fig. S1). The CON spec um, on he o he hand, shows ema kably well dispe sed signals (Fig.2b) wi h peaks om all C′–NH pai s p esen . We hus i s a emp ed he assignmen wi h spec a ha ing he CON spec um as he oo spec um, namely Hα-de ec ed expe i- men s HA(CA)CON, iHA(CA)NCO and (HACA)CON(CA) HA (Män ylah i e al. 2010, 2011). 3D HN-de ec ed expe i- men s HNCACB, CBCA(CO)NH, HNCO and i(HACA) CO(CA)NH (Män ylah i e al. 2009) we e acqui ed o collec HN, C′, Cα and Cβ chemical shi s. Wi h his se o se en 3D spec a, we assigned he majo i y o backbone esonances. Howe e , he e we e se e al ambiguous assignmen s, in pa icula wi hin he a o emen ioned epea ing segmen s. Mo eo e , while p ecise Hα and C′ shi s we e ob ained om he 3D Hα-de ec ed expe imen s and he CON, due o hea y o e lap, Cα/Cβ shi s we e a mo e di icul o ead om he HNCACB and CBCA(CO)NH spec a. Indeed, a p ecise Cα/Cβ chemical shi o 66% o he esidues was ob ained, Fig. 3 a–c Schema ic p esen a ion o magne iza ion ans e pa hway du ing he 4D iHACANCO d, 4D HACACON e and 4D (HACA) CONCAHA expe imen s. Red a ows indica e di ec ans e pa h- way om 1Hα(i) o 15N(i) o 15N(i + 1) whe eas g een a ows indica e a nes ed CαC′ZNZ →NZCα ans e in d) known as he in a esidual il e (Pe mi 2002; B u sche 2002) o highly selec i e CαC′ZNZ →Cα ans e in e). A ows indica e ou -and-back ype magne iza- ion ans e , whe eas one-way a ows ep esen cohe ence ans e ou e which is unidi ec ional. One-le e codes abo e he a ows indi- ca e ime poin s in he pulse sequence. d In a esidual iHACANCO expe imen o co ela e 1Hα(i), 13Cα(i), 13C′(i) and 15N(i) chemical shi s, e he HACACON expe imen , which co ela es chemical shi s o 1Hα(i), 13Cα(i), 13C′(i) and 15N(i + 1) esonances. The (HACA) CONCAHA expe imen o co ela ing 1Hα(i), 13Cα(i), 13C′(i) and 15N(i + 1) esonances. Na ow and wide illed ba s on 1H and 15N channels co espond o ec angula 90° and 180° pulses, espec i ely, applied wi h phase x unless o he wise s a ed. All 13C pulses a e band- selec i e shaped pulses, deno ed by illed na ow ba s (90°) and illed and un illed hal ellipsoids (180°). Un illed ba s a e applied on- eso- nance. The 1H, 15N, 13C′, and 13Cα ca ie posi ions a e 4.7 (wa e ), 118 (cen e o 15N spec al egion), 174ppm (cen e o 13C′ spec al egion), and 56ppm (cen e o 13Cα spec al egion). The 13C ca ie is se ini ially o he middle o 13C′ egion (174ppm), shi ed o 13Cα egion (56ppm) p io o 90° 15N pulse ϕ1 in scheme d). In scheme e) and ), he ca ie is ini ially a 56ppm and shi ed o 174ppm p io o 90° 13C pulse ϕ2, and shi ed back o 56ppm be o e 90° 13C pulse ϕ4. The i s band-selec i e 180° 13C pulse, e ocusing 13Cα magne i- za ion (56ppm, deno ed wi h an as e isk) had du a ion o 788μs a 800MHz. O he band-selec i e 90° and 180° pulses o 13Cα (56ppm) and 13C′ (174 ppm) we e applied wi h du a ions o 240.0 μs and 192.0μs a 800MHz, espec i ely. Band-selec i e 90° and 180° pulses o 13C′/13Cα ha e he shape o Q5 and Q3 (Emsley and Bodenhausen 1992) and du a ion o 240.0μs and 192.0μs a 800MHz, espec i ely. The adiaba ic 180° Chi p b oadband in e sion pulse o in e ing 13Cα and 13C′ magne iza ion in he middle o 1 pe iod had du a ion o 500μs a 800MHz (Böhlen and Bodenhausen 1993). The Wal z-65 sequence (Zhou e al. 2007) wi h s eng h o 4.17kHz was employed o decouple 1H spins. The GARP (Shaka e al. 1985, 1987) wi h ield s eng h o 4.55 kHz was used o decouple 13C du ing acquisi ion. Delay du a ions: τ = 1/(4JHC) ~ 1.7ms; τ2 = 3.4ms (op imized o non- glycine esidues) o 2.2–2.6ms ( o obse ing bo h glycine and non- glycine esidues); ε = du a ion o GH + ield eco e y ~ 0.4ms; 2TC = 1/ (2JCαC′) ~ 9.5 ms; TCA = 1/(6JCαC′) ~ 3.3ms; TA = 1/(4JC′N) ~ 16.6 ms; TC′ = TC + TCC; TCC = 1/(JCαCβ)–1/(4JC′N)–1/(2JCαC′) ~ 0–2.5 ms; TNC ~ 14 ms; TCN ~ 14 ms; TN ~ 14 ms. Maximum 1, 2 and 3 a e es ained in scheme d, 2,max < 2.0*TC′, 3,max < 2.0*TCN, in scheme e, 2,max < 4.0*TA, 3,max < 2.0*TCA, in scheme , 1,max < 2.0*TA, 2,max < 2.0*TNC, 3,max < 2.0*TCN. F equency disc imina ion in 15 N and 13C′ dimensions is ob ained using he S a es-TPPI p o ocol (Ma ion e al. 1989) applied o ϕ1 and ϕ2, espec i ely, whe eas he quad a u e de ec ion in 13Cα dimension is ob ained using he sensi- i i y-enhanced g adien selec ion (Kay e al. 1992; Schleuche e al. 1994). The echo and an iecho signals in 13Cα dimension a e collec ed sepa a ely by in e ing he sign o he GC g adien pulse oge he wi h he in e sion o ψ, espec i ely. Phase cycling: ϕ1 = x, − x; ϕ2 = 2(x), 2(− x); ϕ3 = 4(x), 4(− x); ϕ4 = x; ψ = x; ec. = x, 2(− x), x, − x, 2(x), − x. Selec i e 180° pulse o 13Cα in he middle o delay 2TA induces a Bloch-Siege shi o 13C′ magne iza ion, a ca e ul adjus men o phase (bsp) o he las 13C′ 90° (phase y) pulse is necessa y in scheme d). G adien s eng hs and du a ions: GC = 13 k G/cm (1.6 ms), GH = 13k G/cm (0.4ms). The pulse sequences code and pa ame e ile o B uke A ance sys em a e a ailable om au ho s upon eques ◂ 152 Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 while o Hα, esol ed peaks o 87% o he esidues we e obse ed (Suppl. Fig. S2). To esol e hese ambigui ies and o ex end he numbe o accu a e chemical shi s, we eso ed o 4D NMR spec- oscopy. In o de o bypass he Cα o e lap p oblem in 3D HN-de ec ed expe imen s and o es ablish di ec connec i i- ies be ween Hα and Cα, and o p o ide dispe sion o sol e ambigui ies a ising om occasional o e lap o C′, Hα eso- nances encoun e ed in he 3D Hα-de ec ed expe imen s, we de ised Hα-de ec ed 4D expe imen s wi h Cα as an addi- ional dimension. In addi ion, we de eloped and employed new 4D Hα-s a , HN-de ec expe imen s which can b idge s e ches o e single p olines simila o 3D expe imen s desc ibed in Hellman e al. (2014), bu which ha e wo NH dimensions o enhance peak dispe sion in a sequen ial walk h ough C′, NH and HN, and o p o ide o mo e accu a e NH/ HN chemical shi s han hose ha could be ob ained om he c owded egions o he 1H, 15N HSQC. 4D iHACANCO, HACACON and(HACA)CONCAHA expe imen s The p oposed 4D iHACANCO, HACACON and (HACA) CONCAHA expe imen s a e ex ensions o hei es ablished 3D coun e pa s (Män ylah i e al. 2010, 2011) wi h addi- ional sampling on he ou h 13CA dimension (Fig.3). The cohe ences low h ough he 4D iHACANCO, HACACON and (HACA)CONCAHA expe imen s in Eqs.1, 2, 3: (1) 1 H𝛼(i) 2𝜏 (1 JH𝛼C𝛼 ) ��→ 13C𝛼(i) 2TC (1 JC𝛼N, 1 JC𝛼N, 1 JC𝛼C� ) ��→ 13 C�(i)[2TA− 2;1JC�N,1JC𝛼N,2JC𝛼N] →13C𝛼(i)[2TC�− 2;1JC�𝛼N,2JC𝛼N,1JC𝛼C�]→15N(i)[ 1 ] →13C𝛼(i) [ 2TN− 3;1JC�𝛼N,2JC𝛼N ] 4𝜏(1JH𝛼C𝛼) ��→ 1H𝛼(i)[ 4 ] (2) 1 H𝛼(i) 2𝜏(1JH𝛼C𝛼) ��→ 13C𝛼(i) 2TC(1JC𝛼C�) ��→ 13C�(i) 2TA(1JC�N) ��→ 15 N(i+1)[ 1]→13C�(i)[2TA− 2;1JC�N] →13C𝛼(i) [ 2TC�− 3;1JC𝛼C� ] 4𝜏(1JH𝛼C𝛼) ��→ 1H𝛼(i)[ 4] (3) 1 H𝛼(i−1) 2𝜏(1JH𝛼C𝛼) ��→ 13C𝛼(i−1) 2TC(1JC𝛼C�) ��→ 13 C�(i−1)[2TA− 1;1JC�N] →15N(i)[2TNC − 2;1JC𝛼N,2JC𝛼N,1JC�N] →13C𝛼(i)[2TA− 2;1JC�N] →13C𝛼(i) [ 2TCN − 3;1JC𝛼N,2JC𝛼N ] 4𝜏(1JH𝛼C𝛼) ��→ 1H𝛼(i)[ 4 ] espec i ely. All expe imen s s a wi h he 1Hα(i)→ 13Cα(i) ans e , and he densi y ope a o immedia ely a e he ϕ3 pulse is desc ibed as Hαz(i)Cαz(i) ( ime poin a). Subsequen ly, he magne iza ion is ans e ed o he 13C′ spin ollowed by he labeling o 13C′ chemical shi in 1 o 2. The ele an densi y ope a o a e he ϕ2 pulse in all expe imen s is desc ibed as Cαz(i)C′y(i) ( ime poin b). Nex , he desi ed cohe ence is ans e ed o he 15N spin o he sequen ial esidue in HACACON and (HACA)CONCAHA expe imen s, desc ibed wi h he densi y ope a o Cαz(i)C′z(i) Ny(i + 1) ( ime poin c). In he iHACANCO expe imen , he magne iza ion is solely ans e ed o he 15N spin wi hin he esidue, desc ibed wi h he densi y ope a o Cαz(i)C′z(i) Ny(i) ( ime poin c). A e labeling he 15N chemical shi s in 1 (o 2), he magne iza ion is ans e ed o he 13Cα cohe ence a e he ϕ4 pulse. The ele an densi y ope a o s ( ime poin d) a e Cαy(i)Nz(i) o iHACANCO, Cαy(i)C′z(i) o HACACON and Cαy(i)Nz(i) o (HACA)CONCAHA schemes. The 13Cα chemical shi is labeled du ing he 3 pe iod be ween ime poin s d–e. While he HACACON (Fig.3b) is he con en ional ou -and-back expe imen , he (HACA)CONCAHA and iHACANCO u ilize he in a esidual il e o he selec- i e 13Cα(i)→ 15N(i) ans e (Pe mi 2002; Män ylah i e al. 2010, 2011). Especially in he iHACANCO expe imen , he magne iza ion ans e is nes ed and u he cla i ica ion is deli e ed in he ollowing. A e con e ing magne iza ion o he Hαz(i)Cαz(i) cohe ence ( ime poin a), he 1JCαC′, 1JCαN, 2JCαN and 1JCαCβ couplings a e ac i e du ing he ime in e al (2TC + 2TA + 2TC′ + TCC) = 52–57ms, which con e s i o he Cαz(i)Ny(i) cohe ence ( ime poin c). Howe e , du ing he delay 2TCC, ha can be selec ed o be 0–5ms based on he elaxa ion p ope ies o 13Cα spins, only 1JCαCβ is ac i e. This is o maximize he ans e e iciency du ing he (2TC + 2TA + 2TC′ + TCC) delay. O no e, o a oid chemi- cal shi e olu ion o 13Cα–13C′ mul iple-quan um cohe ence du ing 2TA – 2, an addi ional bu opposi e equency labeling pe iod o 13Cα has been implemen ed in he 2TC′ pe iod. E icien ly, only he chemical shi e olu ion o 13C′ will ake place du ing 2, bu he a ainable esolu ion is limi ed by 2TC′ (= 2TC + TCC) i.e. 2,max is 19–22ms, depending on he se ing o 2TCC (0–5ms). Thus, he 4D iHACANCO, HACACON and (HACA) CONCAHA expe imen s yield co ela ions a ωHA(i), ωCA(i), ωC′(i), ωN(i); ωHA(i), ωCA(i), ωC′(i), ωN(i+1), and ωHA(i), ωCA(i), ωC′(i–1), ωN(i) equencies, espec i ely. Gi en ha equency labeling o 13Cα chemical shi s is implemen ed in a con- s an - ime manne , wi hou leng hening he ac ual pulse sequence and inco po a ing sensi i i y enhanced g adien 153Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 echo in 3, he e is no sensi i i y loss in ol ed in inc eas- ing he dimensionali y o hese Hα-de ec ed expe imen s. Hence, he cohe ence ans e e iciencies p o ided wi h he co esponding 3D expe imen s by Män ylah i e al. (2010, 2011) a e di ec ly compa able o 4D implemen a- ions shown in Fig.3. Indeed, by aking in o accoun ypical alues o one-bond couplings 1JCαC′ = 53Hz, 1JC′N = 15Hz, and 1JCαCβ = 35Hz, and he a e age andom coil alues o one-bond (1JCαN = 10.6Hz) and wo-bond (2JCαN = 7.5Hz) couplings be ween backbone 13Cα and 15N spins (Delaglio e al. 1991), as well as ans e se elaxa ion imes (T2) o 13Cα (= 100ms), 13C′ (= 200ms) and 15N (= 200ms) spins, we can es ima e cohe ence ans e e iciencies o hese expe imen s in IDPs (Män ylah i e al. 2010, 2011). The HACACON is supe io in sensi i i y (I ~ 0.28) in compa i- son o iHACANCO and (HACA)CONCAHA expe imen s, wi h cohe ence ans e e iciencies o 0.22 and 0.18, espec- i ely. Pa icula ly, o he assignmen o p olines, sen- si i i ies o iHACANCO and HACACON a e supe io o he (HACA)CONCAHA scheme, which yields cohe ence ans e o 0.026 o p oline esidues. The sensi i i y loss is associa ed wi h he 15N(i)→ 13Cα(i) ans e , 2TNC, du ing which he 15N magne iza ion is u he modula ed by 1JNCδ coupling in e ac ion in p olines. Figu e4 compa es 3D Hα-de ec ed wi h he new 4D spec- a: p oblems associa ed wi h mul i old o e lap and ambigui- ies in choosing he igh sequen ial connec ion when using 3D spec a can be su passed, and he assignmen p ocedu e expedi ed by ex ending equency labeling o Cα. Wi h he help o hese Hα-de ec ed 4D expe imen s, 76% o he α co ela ions o we e success ully assigned (Suppl. Fig S2). Conside ing ha some N, C′ esonances a e sepa- a ed by less han 0.05ppm in he CON spec um, e.g. co e- sponding esonances wi hin73/113KDAISE78/118 di e only by 0.03–0.04ppm, all peaks we e no expec ed o be esol ed in spec a wi h spec al esolu ions o 0.11 (15N) and 0.05 (13C) ppm. The Hα, Cα shi s we e as ine ec i e in p o iding he needed dispe sion, he smalles peak sepa a ions being com- pa able, e.g. he 76/116Ile alpha peaks di e by only 0.01 and 0.05ppm in 1H and 13C, espec i ely, and hose o 77/117Se e en less (Suppl. Fig. S1). 4D (HACA)CON(CA)NH and(HACA)N(CA)CONH expe imen s Analogously o 4D Hα-de ec ed expe imen s desc ibed abo e, he 4D HN-de ec ed expe imen s (Fig.5), wi h an isola ed p oline assignmen enhancemen , a e based on hei A134Ha-C’-A135N A97Ha-C’-A98N 177.9 178.0 15N 124.9 ppm 15N 124.70 ppm 15N 124.60 ppm 3D HA(CA)CON3D iHA(CA)NCO 3D iHA(CA)NCO 3D iHA(CA)NCO 4D (HACA)CONCAHA 4D (HACA)CONCAHA4D HACACON 4D HACACON 13C (ppm) A135Ha-Ca-N-A134C’ A98Ha-Ca-N-A97C’ 52.5 53.0 A97Ha-Ca-N-E96C’ A94N-A93Ha-Ca-C’ A135N-A134Ha-Ca-C’ A98N-C’A97Ha-Ca- 4.204.25 E96Ha-Ca-A97N C’- 56.5 57.0 57.5 1 H (ppm) 13C (ppm) 1513 N 122.92 ppm,C 177.90 ppm 15 13 N 122.92 ppm,C 177.90 ppm 15 13 N 124.64 ppm,C 176.53 ppm 15 13 N 124.73 ppm,C 176.60 ppm 1H (ppm) 15N 122.92 ppm 4.204.25 4.204.25 4.204.25 4.204.25 177.8 4.30 4.204.25 4.30 4.204.25 4.30 4.154.204.25 Fig. 4 P oblema ics in esonance assignmen wi h 3D Hα-de ec ed expe imen s can be esol ed wi h 4D HACACON and 4D (HACA) CONCAHA expe imen s. The uppe ow o 2D planes om 3D HA(CA)CON and iHA(CA)NCO spec a shows ha he sequen ial walk om 97Ala o 96Glu is ambiguous because he 97Ala Ha, C′ shi pai obse ed in he HA(CA)CON can be ound in se e al planes o he iHA(CA)NCO and is o e lapping wi h o he peaks. While he e is o e lap in he 4D spec a also, he planes a e a mo e easily in e - p e ed and allow o unambiguous assignmen o 98Ala-97Ala-96Glu. G ey labels ma k peaks wi h maximum in adjacen 15N o 13C planes 154 Jou nal o Biomolecula NMR (2020) 74:147–159 1 3 3D coun e pa s (Hellman e al. 2014). Again, sampling o an addi ional 15N dimension can be implemen ed in wi hou in oducing sensi i i y loss hanks o he g adien enhanced cohe ence o de selec i e cohe ence ans e (COS-CT) (Kay e al. 1992; Schleuche e al. 1994). Magne iza ion ans e h ough (HACA)CON(CA)NH and (HACA)N(CA)CONH expe imen s a e b ie ly desc ibed in Eqs.4 and 5, espec i ely: (4) 1 H𝛼(i−1) 2𝜏 (1 JC𝛼H𝛼 ) ��→ 13C𝛼(i−1) 2TC (1 JC𝛼C� ) ��→ 13 C�(i−1)[2TC�N− 1;1JC�N]→15N(i)[ 2] →13C𝛼(i−1) 2TCAN (1JC𝛼N,2JC𝛼N) ��→ 15N(i−1) [ 2TNCA − 3 ;1JC𝛼N,2JC𝛼N ] 4Δ(1JNH ) ��→ 1HN(i−1)[ 4] Like in he Hα-de ec ed expe imen s ( ide sup a), he magne iza ion is i s ans e ed om 1Hα o 13Cα spin ( ime poin a), and u he o ei he 13C′(i–1) spin in (HACA)CON(CA)NH (densi y ope a o Cαz(i–1)C′y(i–1)) o selec i ely o 15N(i) spin in (HACA)N(CA)CONH (densi y ope a o Cαz(i)Ny(i)) a ime poin b. This is ol- lowed by he equency labeling o 13C′(i–1) and 15N(i) chemical shi s du ing 1 in (HACA)CON(CA)NH and (5) 1 H𝛼(i) 2𝜏 (1 JC𝛼H𝛼 ) ��→ 13C𝛼(i) 2TCN (1 JC𝛼N, 2 JC𝛼N ) ��→ 15N(i)[ 1 ] →13C𝛼(i) 2TCAN ,2TC(1JC𝛼N,2JC𝛼N,1JC𝛼C�) ��→ 13C�(i) [ 2TC− 2;1JC𝛼C�]→15N(i+1)[2TNC − 3;1JC�N] 4Δ(1JNH ) ��→ 1HN ( i + 1 )[ 4] ab c d