Recent Advances in Biomolecular NMR Lucia Banci CERM University of - PowerPoint PPT Presentation

Recent Advances in Biomolecular NMR Lucia Banci CERM – University of Florence

Recent Advances in Biomolecular NMR • Protonless NMR for the characterization of Unfolded proteins, Large protein assemblies, Paramagnetic systems • I n cell NMR For studying biomolecules in a cellular context • Combination of Solution and Solid State NMR For characterization of dynamic proteins and large aggregates • Mechanistic Systems Biology To describe and understand biological processes at molecular level

Why protonless NMR? 15 N Inverse (i.e. through 1 H) detection of heteronuclei was a major advanchement!! 13 C Properties of 1 H (high g H , ..) 1 H  high 1 H sensitivity  /  large dipolar interactions  /  efficient relax processes (paramagnetic and large)  relatively low chemical shift dispersion (unfolded systems)

13 C direct detection … with increase in sensitivity, 15 N (high B 0 , cryo!) 13 C direct detection of heteronuclei (low  nuclei) becomes accessible 1 H Isotopic enrichment necessary anyway 13 C direct detection is a complementary tool

13 C direct detection, protonless NMR 15 N A complementary tool for challenging systems 13 C - paramagnetic proteins 1 H - very large proteins - parts of proteins affected by exchange processes - unfolded systems - high salt concentrations

C ´ direct detection – The experiments Set of exclusively heteronuclear experiments based on C ´ and C a detection for sequence specific assignment of a protein More complete information   automation Solution & solid state NMR   common/complementary

C ´ direct detection - IPAP IP AP SUM DIFF Set-up on 13 C- 15 N labeled Alanine Nielsen N.C., Thøgersen H., Sørensen O.W., J. Am. Chem. Soc. , 1995 , 117 , 11365-6 Ottiger M., Delaglio F., Bax A., J. Magn. Reson. , 1998 , 131 , 373-378 Info on the spiltting!! Andersson P., Weigelt J., Otting G., J. Biomol. NMR , 1998 , 12 , 435-441  RDC!!! Duma L., Hediger S., Lesage A., Emsley L., J. Magn. Reson , 2003 , 164 , 187-195 Hu K., Eletsky A. Pervushin K., J. Biomol. NMR , 2003 , 26 , 69 Bertini I., Felli I.C., Kümmerle R., Luchinat C., Pierattelli R., J. Biomol. NMR , 2004 , 30 , 245-251

C ´ direct detection – CON-IPAP CON d( 15 N) C ´ i -N i+1 Transfer pathway: F1(CO)  F3(N,t 1 )  F1(CO,t 2 ) d( 13 C ´ ) Correlations observed: N i -C ´ i-1 CON-IPAP - The delays are:  = 9 ms,  1 = 25 ms,  = t 1 (0). The phase cycle is:  1 = x,-x;  2 = 2x,2(-x);  3 = 4x,4(-x);  IPAP (IP) = x;  IPAP (AP) = - y;  rec = x,(-x),x,(-x),(-x),x,(-x),x. Quadrature detection in the F 1 dimension is obtained by incrementing  1 in a States-TPPI manner.

C ´ direct detection – CON-IPAP CON-IPAP 600 MHz Prototype cryoprobe optimized for 13 C sensitivity (S/N 1400:1) Reduced monomeric SOD (15 kDa) 161 out of the 163 expected correlations are resolved Bermel, W.; Bertini, I.; Felli, I. C.; Kümmerle, R.; Pierattelli, R. J.Magn.Reson. 2006 , 178 , 56-64.

C ´ direct detection – CACO-IPAP CACO d( 13 C a ) C ´ i -C a i Transfer pathway: F1(C a , t 1 )  F1(CO,t 2 ) d( 13 C ´ ) Correlations observed: C a i -C ´ i CACO-IPAP - The delays are:  = 9 ms. The phase cycle is:  IPAP (IP)= x,-x and  rec = x,-x;  IPAP (AP)= -y, y and  rec = x, -x. Quadrature detection in the F 1 dimension is obtained by incrementing  1 in a States-TPPI manner.

C ´ direct detection – CBCACO-IPAP CBCACO C ´ i -C b i d( 13 C a,b ) d( 13 C a ) C ´ i -C a i Transfer pathway: F1(C a / b , t 1 )  F1(C a , t 2 )  F1(CO,t 3 ) Correlations observed: C b i - C a i -C ´ i , C a i - C a i -C ´ i d( 13 C ´ ) CBCACO-IPAP - The delays are:  = 9 ms,  1 = 8 ms. The phase cycle is:  1 = x,-x;  2 = 8x,8(-x);  3 = 2y,2(-y);  IPAP (IP) = 4(x),4(-x);  IPAP (AP) = 4(-y),4(y);  rec = x,(-x),(-x),x,(-x),x,x,(-x). Quadrature detection in the F 1 and F 2 dimensions is obtained by incrementing  1 and  3 in a States-TPPI manner.

C ´ direct detection – S 3 E CCCO C ´ i -C d i d( 13 C a,b,.. ) C ´ i -C  i d( 13 C a,b ) d( 13 C) Transfer pathway: F1(C ali , t 1 )  F1(C a , t 2 )  F1(CO,t 3 ) i - C a i -C ´ i , C a i - C a i -C ´ i Correlations observed: C ali C ´ i -C b i d( 13 C ´ ) CCCO-IPAP - The delays are:  = 9ms,  = t 1 (0). The phase cycle is:  1 = x, -x;  2 = 2x, 2(-x);  IPAP (IP) = 4x, 4(-x);  IPAP (AP) = 4(-y),4y;  rec = x, (- x), (-x), x, (-x), x, x, (-x). Quadrature detection in the F 1 and F 2 dimensions is obtained by incrementing  1 and  2 respectively in a States-TPPI manner.

C ´ detection - Assignment strategy CCCON C ´ i -C d i CBCACON d( 13 C a,b,.. ) C ´ i -C b C ´ i -C  i i CACON d( 13 C a,b ) d( 13 C) d( 13 C a ) C ´ i -C a C ´ i -C a C ´ i -C b i i i Spin system identification d( 13 C ´ ) d( 13 C ´ ) d( 13 C ´ )

CACO, CBCACO, CCCO-IPAP CACO-IPAP CBCACO-IPAP CCCO-IPAP 600 MHz Cryoprobe optimized for 13 C sensitivity (S/N 1400:1) 16 scans 2-3.5 hours the majority of the 13 C spin systems could be assigned Bermel W., Bertini I., Duma L., Felli I.C., Emsley L., Pierattelli R., Vasos P.R., Angew. Chem. , 2005 , 44 , 3089- 3092 Bermel, W., Bertini, I., Felli, I. C., Kümmerle, R., Pierattelli, R. J.Magn.Reson. 2006 , 178 , 56-64.

C ´ detection - Assignment strategy CBCANCO C ´ i -C b i+1 C ´ i -C b COCON i d( 13 C a,b ) C ´ i -C ´ i+1 CANCO C ´ i -C a C ´ i -C a i+1 i+1 d( 13 C ´ ) C ´ i -C ´ i d( 13 C a ) C ´ i -C a C ´ i -C a C ´ i -C ´ i-1 i i Sequential assignment d( 13 C ´ ) d( 13 C ´ ) d( 13 C ´ )

C ´ detection - Assignment strategy CBCACON-IPAP CCCON-IPAP D d ( 13 C’) D d ( 13 C’) @600 MHz CPTXO (S/N 1400:1) on 1.5 mM 13 C, 15 N labeled reduced monomeric SOD. CBCACON-IPAP, 16 scans, 3 days, CCCON-IPAP, 32 scans, 4.5 days. 96 % of the 13 C resonances could be identified Bermel W., Bertini I., Felli I.C., Kümmerle R., Pierattelli R., JMR , 2006 , 178 , 56-64

C ´ detection - Assignment strategy PRO 74 LYS 75 Bermel, W., Bertini, I., Felli, I. C., Kümmerle, R., Pierattelli, R. J.Magn.Reson. 2006 , 178 , 56-64.

One of the powerful applications of 13 C direct detection NMR Intrinsically disordered proteins - IDPs! Aggregated Folded

... Reduction in 1 H chemical shifts Cu(I)Zn(II)SOD 153 AA Synuclein 140 AA Well folded IDP

13 C carbonyl direct detection – IDPs 105 105 110 110 115 115 15 N chemical shift 120 15 N chemical shift 120 125 125 130 130 135 135 C ´ i-1 -N i H N 140 i -N i 140 145 145 9,2 9,0 8,8 8,6 8,4 8,2 8,0 7,8 7,6 7,4 178 177 176 175 174 173 172 171 170 1 H chemical shift 13C chemical shift Zhang, H., Neal, S., Wishart, D.S., J. Biomol. NMR 2003 , 25 , 173-195 Schwarzinger S., Kroon G.J., Foss T.R., Chung J., Wright P.E., Dyson H.J., J. Am. Chem. Soc. 2001 , 123 , 2970-2978

CON of intrinsically unfolded a -synyclein All residues assigned (N,C ´ ,C a ,C b ) Prolines are visible Bermel W., Bertini I., Felli I.C., Lee Y.M., Luchinat C., Pierattelli R., J. Am. Chem. Soc. , 2006 , 128 , 3918-3919

Intrinsically unfolded a -synyclein Strips from the 3D COCON-IPAP Sequence specific assignment 3D CBCACON-IPAP 3D COCON-IPAP Bermel W., Bertini I., Felli I.C., Lee Y.M., Luchinat C., Pierattelli R ., J. Am. Chem. Soc. , 2006 , 128 , 3918-3919

Securin – Intrinsically disordered protein Interphase Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis Securin inhibitor of separase Metaphase Anaphase Securin Intrinsically disordered protein (IDP!) 202 AA (>10% PROs)

Intrinsically unfolded human securin Securin – 202 AA, 24 PRO GLY (N) GLY (N) 9 corr obs 11 corr obs PRO (N) Observed well resolved peaks: 22 corr obs CON: 165 HSQC: 122 82% of the expected 68% of the expected 82% of the whole protein 60% of the whole protein Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009 , 130, 16873-16879

Intrinsically unfolded human securin Securin – 202 AA, 24 PRO Correlations observed: C a i ,C ´ i ,N i+1 C b i , C ´ i ,N i+1 193, out of the 201 expected, spin patterns are identified (96%) in CBCACON-IPAP. Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009 , 130, 16873-16879

Assignment and chemical shift analysis of securin a -helical secondary structure propensity for the stretch D 150 -F 159 Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009 , 130, 16873-16879

Human securin - other NMR observables D 150 -F 159 , E 113 -S 127 and W 174 -L 178 Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009 , 130, 16873-16879

13 C direct detection – Speeding up?  Can one implement all the tricks to reduce experimental time? -Longitudinal relaxation enhancement Decrease the recycle delay -Reduction in datapoints acquired in indirect dimensions

13 C direct detection – Speeding up Longitudinal relaxation enhancement 1 H-start, 1 H-flip Diercks, T.; Daniels, M.; Kaptein, R. J.Biomol.NMR 2005 , 33 , 243-259. Deschamps, M.; Campbell, I. D. J.Magn Reson. 2006 , 178 , 206-211. Schanda, P.; Brutscher, B. J.Am.Chem.Soc. 2005 , 127 , 8014-8015. Müller, L. J.Biomol.NMR 2008 , 42 , 129-137. Bermel, W., Bertini I., Felli I.C., Pierattelli, R., J. Am. Chem. Soc. , 2009 , 131, 15339-15345