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 (large and paramagnetic molecules)  relatively low chemical shift dispersion (unfolded systems)

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

13 C direct detection – unique properties Different spins, different properties! 1 H D Dn ( 1 H) D d ( 1 H) D d ( 1 H) 13 C D Dn ( 13 C) D d ( 13 C) D d ( 13 C)

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

One of the powerful applications of 13 C direct detection NMR Intrinsically disordered proteins - IDPs! Aggregated Folded By I. Felli & R. Pierattelli

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

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 – increasing dimensions nD? High resolution necessary for IDPs only possible through reduced sampling strategies & longitudinal relaxation enhancement

13 C direct detection – 4Ds C’ (i) -N (i+1) C α (i) -N (i+1) C α (i) -N (i) 4D (H N -flip)NCACON - 1d, 12h, 0.036 % 4 scans per increment, 0.7 s d1, 2000 points, max evolution: 40 ms ( 15 N), 24 ms ( 13 C), 24 ms ( 15 N) Bermel W., Bertini I., Felli I.C., Gonnelli L., Koźmiński W., Piai A., Pierattelli R., Stanek J., J. Biomol. NMR , 2012 , 53 , 293-301.

13 C direct detection – 4D HCBCACON C’ (i) -N (i+1) H α , β (i) -C α , β (i) H ali (i) -C ali (i) 4D HCBCACON – 1d, 4h, 0.053 % 4 scans per increment, 1.1 s d1, 1200 points, max evolution: 30 ms ( 15 N), 6 ms ( 13 C), 10 ms ( 1 H) 4D HCCCON - 1d, 19h, 0.015 % 4 scans per increment, 1.1 s d1, 1800 points, max evolution: 28 ms ( 15 N), 12 ms ( 13 C), 20 ms ( 1 H) Bermel W., Bertini I., Felli I.C., Gonnelli L., Koźmiński W., Piai A., Pierattelli R., Stanek J., J. Biomol. NMR , 2012 , 53 , 293-301.

In-cell NMR spectroscopy

In-cell NMR spectroscopy • In-cell NMR allows the characterization of biomolecules inside living cells. • It relies on high resolution NMR experiments to obtain information at atomic resolution on biomolecule structure, folding and interactions. • It has a high biological relevance, as the biomolecules are monitored in a cellular environment.

Different living organisms • Different cells have been and are used: bacteria, oocytes and mammalian cells. Different techniques are exploited to obtain high protein concentration: overexpression and injection/insertion. • Prokaryotic cells are more commonly used. Indeed, the bacterial cytoplasm is a good model of the eukaryotic one, in terms of molecular crowding, pH and redox potential. • Eukaryotic cells have been used to monitor protein interactions with specific cellular components, such as kinases. They have the machineries and chaperones for the correct maturation of eukaryotic proteins.

Protein insertion in eukaryotic cells Proteins are produced in bacteria and then inserted in mammalian cells strains (e.g. CHO, HeLa) or in Xenopus laevis oocytes. For mammalian cell cultures, protein To insert the protein into X. laevis oocytes insertion is achieved by using cell- the microinjection technique is used. penetrating peptides to deliver a fusion protein, or porins to permeabilize the cells. Fig. From: D.S. Burz, A. Shekhtman, Nature, 458 (2009). K. Inomata et al , Nature, 458 (2009). P. Selenko, G. Wagner, J Struct Biol, 158 (2007).

NMR pulse sequences Virtually any solution NMR pulse sequence can be used for in-cell NMR experiments, BUT: • The low sensitivity of NMR requires high protein concentration, not always obtained; • The viability of the cell sample is limited to few hours; Therefore fast and sensitive experiments are often needed: • Fast pulsing experiments: 2D SOFAST-HMQC, 3D BEST-triple resonance experiments; • Sparsed sampling experiments: non-uniform sampling, projection spectroscopy.

SOFAST-HMQC The 1 H- 15 N SOFAST-HMQC (1) is often used for in-cell NMR. It is the fast equivalent of the 1 H- 15 N HMQC. The selective 1 H pulse excites only the amide protons, allowing faster longitudinal relaxation between the scans: shorter interscan delays. The pulse can be set at the Ernst angle α (120° instead of 90 ° ), to maximize sensitivity. Schanda,P., Kupce,E., and Brutscher,B., J. Biomol. NMR 33 , 199-211 (2005).

A functional process analyzed in living cells with atomic details: Maturation of Cu,Zn-SOD1

A Cu transport process in human cells Cu(II) Cu(I) Regulators CCS SOD Cu(I) Ctr Nucleus MT No free copper ions in the cytoplasm

Superoxide Dismutase Present in cytoplasm, mitochondrial IMS, nucleus, peroxisomes It catalyzes the dismutation of superoxide anion through reduction and oxidation of the copper ion - + 2H +  O 2 + H 2 O 2 ) ‏ (2O 2 Quaternary Structure -Dimeric protein A conserved SS bond Cu Two metal ions per subunit Zn

hSOD1 maturation C57 Cu Zinc Copper binding binding Zn C146 monomeric apo hSOD1 SH-SH Disulfide bond formation SS bond dimeric (Cu 2 ,Zn 2 ) hSOD1 SS These post translational modifications affect the fold properties and monomer/dimer equilibrium

CCS – the chaperon required for SOD1 in vivo maturation Domain II SOD1-like Domain I Atx1-like Domain III Disordered It dimerizes through the SOD1-like domain

How is SOD1 in the cytoplasm? Maturation of SOD1 in human cells followed by in-cell NMR Zinc uptake is very selective in intact cells at variance with cell lysates and in vitro Red: no metal added Blue: Zn(II) added Banci, L., Barbieri, L., Bertini, I., Cantini, F., Luchinat, E., PlosONE 2011 Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Zhao, Y., Aricescu A.R., submitted, 2012

15 N-Cys labelling: cysteine redox state 15 N Cys 146 E,Zn-SOD1 SH-SH Cys 111 Cys 6 E,Zn-SOD1 SH-SH 1 H All cysteines of E,Zn-SOD1 are reduced. Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Aricescu A.R., submitted, 2012