Biomolecular NMR. K.V.R. Chary Workshop on NMR and it s applications in Biological Systems November 24, 2009

Transcription

1 Biomolecular NMR K.V.R. Chary Workshop on NMR and it s applications in Biological Systems TIFR November 24, 2009

2 RECAP

3 Vector representation of bulk magnetization Z Z MZ MY Y Z X X MX X Y Y

4 Evolution of spin operator under a pulse (π/2)x Pulse: X Η 1 Iz -Iy Iy -Iz

5 Evolution of spin operator under a pulse (π/2)y Pulse: Y Η 1 Iz Ix -Ix -Iz

6 Evolution of spin operator under a pulse βy Iz Iz cos(β) + Ix sin(β) βx Iz Iz cos(β) Iy sin(β)

7 Evolution of spin operator under a pulse βy Iz Iz cos(β) + Ix sin(β) (π/2)y Iz Ix (π)y Iz -Iz

8 Evolution of spin operator under a pulse βx Iz Iz cos(β) - Iy sin(β) (π/2)x Iz -Iy (π)x Iz -Iz

9 Evolution under Chemical Shift (Hδ = ΩΗIz) Y t1 My Sin (Ωt) Mx Cos (Ωt) Y Ωt Mx X X

10 Evolution under Chemical Shift (Hδ = ΩΗIz) Rotation about the z-axis I I cos(ωt) + I sin(ωt) 1x 1x 1y I I cos(ωt) I sin(ωt) 1y 1y 1x I I 1z 1z

11 Evolution under Chemical Shift (Hδ = ΩΙIz) X/Y Magnetization : Η 1 Ix y Hδ= ΩΙIz Iy Angle = ΩΙ t -Iy -Ix

12 Spin-echo pulse sequence (π/2)φ (π)ϕ τ τ 2τ = 1/2J τ is the refocussing delay. t

13 Spin-echo pulse sequence Y Y X τ b Ωt Y a X πx Y X Ωt b τ X a Effect of a SE sequence on an uncoupled spin with precessional frequency Ω.

14 Spin-echo pulse sequence Spin-echo pulse sequence removes the dephasing caused by the field inhomogenity. Other processes which contribute to the decay of transverse magnetization continue to exert their influence. How about in the case of coupled spin-systems?

15 Coupled Spin Transformations CHCl2-CHO A X

16 AX spin system αx ΩA+π(JAX/2) βx ΩA π(jax/2) αa ΩX+π(JAX/2) βa ΩX π(jax/2) NMR Spectrum of an AX spin system. Offset frequencies are depicted below the individual lines. The spin states (α and β) of the coupled partner associated with individual lines are depicted on top of the spectrum.

17 Spin-echo pulse sequence In the case of coupled spin-systems The spin-echo sequence refocuses the chemical shifts. It phase modulates the J-couplings that may be present.

18 Spin-echo pulse sequence Effect of (π)x pulse Tilts the magnetization vectors with respect to x-axis. Swaps the associated spin states of the coupled partner.

19 Spin-echo pulse sequence Y Y X τ αx Y βx X Y Y ( π x)a X αx ( π x)x βx X βx τ αx Effect on a J-coupled spin A. The αx and β x represent the spin states of the coupling partner spin X. αx X βx

20 Evolution under Couplings (HJ = 2πJ12I1zI2z) I1x I1x cos(πj12t) + 2I1yI2z sin(πj12t) I1y I1y cos(πj12t) 2I1xI2z sin(πj12t) 2I1xI2z 2I1xI2z cos(πj12t) + I1y sin(πj12t) 2I1yI2z 2I1yI2z cos(πj12t) I1x sin(πj12t)

21 Evolution under Couplings (HJ = 2πJ12I1zI2z) X Magnetization : y Η 1 I1x HJ=2πJ12I1zI2z 2I1yI2z Angle =2πJ12t -2I1yI2z -I1x

22 Evolution under Couplings (HJ = 2πJ12I1zI2z) Y Magnetization : x Η 1 I1y HJ=2πJ12I1zI2z 2I1xI2z Angle =2πJ12t -2I1xI2z -I1y

23 Vector repre tion of in-phase and antiphase magnetization Z Z I1Y I1X Y Y Z X X 2I1xI2z X Y

24 Vector repre tion of in-phase and antiphase magnetization Z Z 2I1YI2Z I1X Y Y Z X X 2I1XI2Z X Y

25 Spin-echo pulse sequence (π/2)φ (π)ϕ I S τ (π/2)φ 5 τ t (π)ϕ (π/2)φ I S 4 (π)ϕ I τ 4 S 5 τ t τ 4 5 τ t

26 Biomolecular Structure K.V.R. Chary Workshop on NMR and it s applications in Biological Systems TIFR November 24, 2009

27 It is in the structure Understanding the structure is important for understanding both biology and chemistry.

28 The water molecule

29 The benzene molecule (A flat Hexagon; 1929) Kathleen Yardley Lonsdale( ) was a crystallographer who established the chemical structure of benzene. She was also the first woman to become a Fellow of the Royal Society.

30 The three important biomolecules DNA, RNA and Proteins

31 The DNA A string of G A G C G Nucleotide pearls C A T G C DNA consists of four different pearls, nucleotides: adenine (A), cytosine (C), guanine (G) and thymine (T).

32 The DNA Too simple a carrier for hereditary traits!!!!! Francis Crick s first sketch The original DNA Model designed by W & C

33 The RNA DNA cousin Another string of G A G C G Nucleotide pearls U A U G C At the beginning of the 1950s, scientists realized that most of the RNA is found in small particles in the cytoplasm, however, consists four different pearls, nucleotides: adenine (A), cytosine (C), guanine (G) and uracil (U).

34 The RNA DNA cousin Another string of Nucleotide pearls At the beginning of the 1950s, scientists realized that most of the RNA is found in small particles in the cytoplasm, however, consists four different pearls, nucleotides: adenine (A), cytosine (C), guanine (G) and uracil (U).

35 The RNA DNA cousin Another string of Nucleotide pearls At the beginning of the 1950s, scientists realized that most of the RNA is found in small particles in the cytoplasm, however, consists four different pearls, nucleotides: adenine (A), cytosine (C), guanine (G) and uracil (U).

36 Proteins A string of T I F R H amino acid pearls H R T I F Proteins, however, consists of twenty different pearls, amino acid : A C D E F G H I K L M N P Q R S T V W Y (thought to be genetic material!!!)

37 Primary structure ADQLTEEQIAEFKEAFSLFDKDGD GTITTKELGTVMRSLGQNPTEAELQDMIN EVDADGNGTIDFPEFLTMMARKMKDTD SEEEIREAFRVFDKDGNGYISAAELRHVM TNLGEKLTDEEVDEMIREADIDGDGQVN To date, the largest protein known is titin (Mr ~ 3000 kda; YEEFVQMMTAK 26,926 residues), which is found in skeletal and cardiac muscle.

38 Secondary structure α helical & β - sheet structures

39 A B C D E Secondary structural elements in proteins (A) α-helix, (B) anti-parallel b-sheet, (C) parallel b-sheet (D) Type I b-turn and (E) Type II b-turn.

40 Tertiary structure Calcium Binding Protein Tertiary structure describes the packing and the fold of secondary structural elements and amino acid side-chains resulting into a compact 3D structure.

41 Tertiary structure Haemoglobin Quaternary structure is determined by the association of more than one polypeptide chain to form multimeric proteins, as is the case in haemoglobin, which contains four subunits.

42 The hemolglobin molecule Perutz and Kendrew 1962 Max Perutz, 1978 Hemoglobin is a tetramer composed of 4 globin molecules; 2 alpha globins and 2 beta globins. The alpha globin chain is composed of 141 amino acids and the beta globin chain is composed of 146 amino acids (Perutz, 1978). Both alpha and beta globin proteins share similar secondary and tertiary structures, each with 8 helical segments (labeled helix A-G) (Keates, 2004).

43 The ribosome Cellular Nobel molecule factory

44 800 kda 20 proteins 1 rrna (1600 nts) (Ramakrishnan and collaborators 2000).

45 1500 kda 33 proteins 2 rrna (2900 & 120 nts) (Yonath and collaborators 2000).

46 Proteins With the completion of the Human Genome Project, the emphasis is shifting to the protein compliment of the human organism. This has given rise to the science of proteomics, the study of all the proteins produced by cell type and organism.

47 Proteins The term proteome refers to all the proteins expressed by a genome, and it involves the identification of proteins in the body and the determination of their role in physiological and pathophysiological functions. While a genome remains unchanged to a large extent, the proteins in any particular cell change dramatically as genes are turned on and off in response to its environment.

48 Proteins The importance of the proteome cannot be overstated as it is the proteins within the cell that provide structure, produce energy, and allow communication, movement and reproduction. Basically, proteins provide structural and functional framework for cellular life. Genetic information is static while the protein complement of a cell is dynamic.

49 IT is not easy to study proteins Proteins cannot be amplified like DNA, therefore less abundant sequences are more difficult to detect. Proteins have secondary and tertiary structure that must often be maintained during their analysis. Proteins can be denatured by the action of enzymes, heat, light or by aggressive mixing as in beating egg whites. Some proteins are difficult to analyze due to their poor solubility.

50 In this post genomic era This revolution in genomics has provided extensive databases of newly discovered proteins. Deciphering the functions of these myriad proteins poses a major intellectual challenge. Do these primary sequences provide any insight into the protein function?

51 Too many sequences, too little time The average protein is about 100 amino acids long. There are 20 different amino acids which could be put at each position. Thus there are 20 raised to the 100th power or about 10,000,000,000,000,000,000,000,000,000,000,000,000,000,0 00,000,000,000,000,000,000,000,000,000,000,000,000,000,0 00,000,000,000,000,000,000,000,000,000,000,000,000,000,0 00,000 possible 100 amino acid proteins.

52 Too many sequences, too little time On the one hand, this suggests that almost any imaginable protein structure is possible. On the other hand, finding the sequence we want in such a large number of possibilities will require more than just luck. Fortunately, nature has provided us with an abundance of examples to use as starting points in our design. Thus far, most of the engineered proteins made have just been slightly modified versions of the natural ones. The next step is to take parts of different natural devices and combine them to create new functions and control capabilities.

53 What is essential to understand a protein function? Experimental determination of conformation of the active site disposition of solvent molecules amplitudes of molecular motions bringing functional groups into close proximity

54 Structural biologists so far have focused mainly on the details of protein function, asking the question (at the`end' of the biological discovery process). `How does the protein achieve its function? Can we now use protein structure to answer the more basic question `What does the protein do?'

55 In fact, the structural studies should now be gainfully employed as part of early phase of biological discovery, to answer the question `what is the function of this protein?' This is the defining feature of Structure-based functional proteomics

56 Haemoglobin Myoglobin? Muscle specific cdna No function known 1HCO.pdb 1A6G.pdb BLAST search with this look cdna alike does not!!! elicit a Haemoglobin hit They Sequence homology = 24.5% The latter may have the same function as Haemoglobin

57 Eh.? CaBP Calmodulin cdna from E.histolytica No function known 1 2 1CFC: ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEI--REA Eh.CABP: 1CFC: MAEALFKEIDVNGDGAVSYEEVKAFVSKKRAIKNEQLLQLIFKSIDADGNGEIDQNEFAKFYGSIQGQDLSDDKIGLKVL FRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK Eh.CABP: YKLMDVDGDGKLTKEEV----TSFFKKHGIEKVAEQVMKADANGDGYITLEEFLEFSL 3 4 BLAST search with this cdna elicited a Calmodulin hit with They alike resemblance factor 138;look Reason: Loop!!! Homology Sequence homology = 28%

58 Advantages of 3D data over the sequence data: Firstly, 3D data can provide new insights into biology by elucidating relatedness to proteins of known biological function, which could not have been arrived at by sequence analysis alone. Secondly, structural information can identify binding motifs and catalytic centers even for proteins without a known biological function. These are the two main principles of The structure-based functional proteomics approach.

59 Challenge To structurally characterize the proteome there is a need for many high resolution structures of proteins & for detailed description of their dynamics.

60 Impact will be on Fundamental Biology, Medicine and Biotechnology. Structures will be used to understand the molecular basis of disease, to develop diagnostics, for drug design and in enzyme engineering.

61 Over the next few years The ratio of known protein sequences to known protein 3D structures will increase dramatically. Homology modelling may reduce the size of the problem. But, this will not yield high resolution models of structures and dynamics.

62 Although the number of crystal and NMR structures deposited each year in the PDB has grown exponentially, the number of structures unrelated to other proteins has not. Why is it so? This is due in part to the practice of structural biologists looking ever deeper at biological problems, and thus studying complexes, mutants, and relatives of proteins of already known structure.

63 Also, the universe of protein structures is definitely finite, and chance occurrences of structural similarity have become increasingly common. This trend would appear to be strongly positive for the task of deriving function from these structures.

64 The message The rate of generation of new structures ought to accelerate, to even start keeping up with the sequence information being generated now.

65

66 Linus C. Pauling Robert B. Corey Frederick Sanger G.N.Ramachandran Max F. Perutz John C. Kendrew Some eminent scientists involved in protein structure determination.

67 5000 A B 500 Number of NMR Structures Number of Structures X-Ray NMR Year Molecular Weight (kda) 40 50

68 Conformation of Biological Molecules

69 Ri+1 Ri Polymer B φi H φ i+1 φi Polymethylene Polysaccharide H B Ri+2 H φ i+2 C C C H H H H ψi O Glc φ i+1 ψ i+1 {θ }={φ i} φ i+2 O Glc ψi φi Cα N H Ri+1 O N Cα C ωi C {θ }={φ i, ψ i, ω i, χ i} χi H Ri O H H5 O Polynucleotide αi P5 O5 βi C5 H4 γi H3 δ i/ν 4 i C4 H5 O4 {θ }={α i, βi, γ i, δ i, ε i, ξ i, χ i, ν i} C3 O εi O3 ν 0i ν 3i O {θ }={φ i, ψ i} Glc H Polypeptide {θ }={φ i} H C φi B φ i+1 C2 C1 ν 2i χi ν 1i ξi H2 P3 O H2 H1 Ri Schematic representation of linear polymers. B represents backbone, R i are the side chains and {θi} is a set of torsion angles which determine the conformation of the ith unit.

70 The torsion angle D A D φ B C A φ D -φ B(C) A B(C) The Newman projections and definition of torsional angles for a four-atom molecular segment, A-B-C-D. The 3D structure of such a molecule is determined by the three bond lengths, two bond angles and one torsional angle In general, to define the 3D geometry for a linear molecules containing N atoms, one needs N-1 bond-lengths, N-2 bond angles and N-3 torsional anlges, a total of 3N-6 structural elements.

71 25 CH3 CH3 H 20 Potential energy (kj/mole) CH3 H CH3 H H CH3 H 15 H H H H H H H CH g+ 5 g t Dihedral angle (in degrees) Potential energy of n-butane as a function of dihedral angle around the central C-C bond.

72 tg4 6 φ (in degrees) g+ t tt g-t tg D iso-energy map of n-pentane showing low energy conformers for a hydrocarbon chain.

73 Typical conformations in biopolymers

74 H H ψ φi N H χi ωi i Cα C' Ri O N {θ }={φ i, ψ i, ω i, χ i} Backbone (φ, ψ, ω ) and side-chain (χ1) torsion angles in an amino acid residue. The π electron in the C=O bond are delocalised giving a partial double bond character to the C -N bond and thus restricting the angle ωι to a trans conformation.

75 C'i-1 φi C'i Cα HN α Ni+1 Ni ψi Cβ Cα i+1 C ωi C' Hα φ i=c'i-1-ni-cα i -C'i Hα Hβ i O Cβ ψ i=ni-cα i-c'i-ni+1 O Hα HN χ i1 Ni+1 HN ω i=cα i-c'i-ni+1-c'i+1 2 Cβ Cγ Hβ 3 C' χ i1=ni-cα i-cβ i-cγ i Newman projection diagram describing backbone (φ, ψ, ω ) and the side-chain (χ1) torsion angles in amino acid residues with a methylene Cβ group.

76 Proteins The peptide bond A resonance structure is invoked to explain the rigidity of the peptide group. Optimal electron delocalization (πbonding) within the peptide group depends upon the planar arrangement of these atoms. The partial double-bond character of the peptide bond is reflected in the intermediate length of the peptide ( Å) bond relative to single (1.49 Å) and double (1.27 Å) carbonnitrogen bonds.

77 Proteins The peptide bond Trans-Cis The steric strain due to the closer approach of the Cα positions in the cis configuration makes it less favorable.

78 Ramachandran plot for EhCaBP

79 Abbreviation and numbering for atoms in amino acids. IUPAC, IUBMB, IUPAB

80 Average 1H, 13C and 15N chemical shifts with standard deviations for amino acid residues obtained from BMRB. δ (ppm) Spin ALA HN 8.18±0.63C ±2.62 Hα 4.26±0.45Cα 53.19±2.11 Hβ 1.36±0.28Cβ 19.04±2.48 ARG Spin δ (ppm) Residue N ±4.54 HN 8.23±0.62C ±3.96 Hα 4.29±0.48Cα 56.84±2.52 Hβ2 1.80±0.30Cβ 30.71±2.46 Hβ3 1.78±0.29Cγ 27.34±2.35 Hγ2 1.58±0.28Cδ 43.20±2.05 Hγ3 1.56±0.30Cζ ±2.17 Hδ2 3.11±0.29N ±3.97 Hδ3 3.11±0.27Nε 93.21±15.12

81 The protein sample preparation

82 Other alternatives Use of NMR active nuclei such as 13C and 15N which have: Large coupling with 1H ( Hz) Large dispersion in chemical shifts But these nuclei have low natural abundance 13 C = 1 : N = 1 : 300 Need for isotope labeling

83 Biosynthetic overexpression of the protein

84 Isotope labeling The host micro-organism, overexpressing the protein of interest, is grown on minimal media containing [13C6] glucose as the sole source of carbon or/and [15N] labeled ammonium chloride as the sole source of nitrogen. Fractionally or Uniformly!!!

85 15 N labeling results in HN 1 N Hα Cα R O 12 C 1 HN N 15 Hα 1 12 Cα O 12 C R Record 2D [15N-1H] HSQC experiment. No. of Peaks = No. of amide 15N-1H pairs ~ No. of amino acid residues present in the protein-no of prolines Experimental time ~ 10 mins (with 1mM sample)

86 2D 15N-1H HSQC 2D HSQC

87 Protein Engineering for NMR studies Use of as many NMR active nuclei (such as 1H, 2H, 13C and 15N) as possible/required probes. Uniform or selective isotope (13C/15N) labeling or unlabeling of proteins.

88 and.. 2H labeling Stereo-Array isotope labelling (SAIL) Splicing protein fragments so that only labeled domains in a natural abundance background can be characterized.

89 Optimization of experimental conditions Temperature ph