Transcript of NMR in biology: Structure, dynamics and energetics Gaya Amarasinghe, Ph.D. Department of Pathology...
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- NMR in biology: Structure, dynamics and energetics Gaya
Amarasinghe, Ph.D. Department of Pathology and Immunology
gamarasinghe@path.wustl.edu CSRB 7752
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- NMR? Nuclear Magnetic Resonance Spectroscopy Today, we will
look at how NMR can provide insight in to biological
macromolecules. This information often compliment those obtained
from other structural methods.
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- http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html
/1dnmr.htm NMR Spectra contains a lot of useful information: from
small molecule to macromolecule.
http://www.nature.com/nature/journal/v418/n6894/fig_tab/
nature00860_F1.html Few peaks Sharper lines Overall very easy to
interpret Many peaks Broader lines Overall NOT very easy to
interpret
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- Structure determination by NMR NMR relaxation how to look at
molecular motion (dynamics by NMR) Ligand binding by NMR
Energetics
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- Outline for Bio 5068 December 10 Why study NMR (general
discussion) 1.What is the NMR signal (some theory) 2.What
information can you get from NMR (structure, dynamics, and
energetic from chemical shifts, coupling (spin and dipolar),
relaxation) 3.What are the differences between signal from NMR vs
x-ray crystallography (we will come back to this after going
through how to determine structures by NMR) Practical aspects of
NMR 1.instrumentation 2.Sample signal vs water signal 3.Sample
preparation (very basic aspects & deal with specific labeling
during the description of experiments) Assignments and structure
determination 1.2-D experiments 2.3/4-D experiments 3.Restraints
and structure calculations Assessing quality of structures 1.NMR
structure quality assessment 2.Comparison with x-ray
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- Nuclear transitions Rotational transitions Translational
transitions Electronic transitions Diffractions NMR works in the rf
range- after absorption of energy by nuclei, dissipation of energy
and the time it takes Reveals information about the conformation
and structure. For diffraction, the limit of resolution is
wavelength!!
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- Protein Structures from an NMR Perspective Background We are
using NMR Information to FOLD the Protein. We need to know how this
NMR data relates to a protein structure. We need to know the
specific details of properly folded protein structures to verify
the accuracy of our own structures. We need to know how to
determine what NMR experiments are required. We need to know how to
use the NMR data to calculate a protein structure. We need to know
how to use the protein structure to understand biological
function
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- Protein Structures from an NMR Perspective Distance from
Correct Structure NMR Data Analysis Correct structure X Not A
Direct Path! Interpreting NMR Data Requires Making Informed Guesses
to Move Toward the Correct Fold Initial rapid convergence to
approximate correct fold Iterative guesses allow correct fold to
emerge Analyzing NMR Data is a Non-Trivial Task! there is an
abundance of data that needs to be interpreted
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- Current PDB statistics (as of 3/27/2012) Exp.Met hod Proteins
Nucleic Acids Protein/Nucleic Acid Complexes Total
X-RAY658281346326070436 NMR81679751869335 ratio8.061.3817.53
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- Nuclei are positively charged many have a spin associated with
them. Moving chargeproduces a magnetic field that has a magnetic
moment Spin angular moment
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- MassChargeI Even I=0 EvenOdd I= integer OddI=half integer
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- How do we detect the NMR signal?
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- Practical aspects of NMR 1.instrumentation 2.Sample signal vs
water signal 3.Sample preparation (very basic aspects & deal
with specific labeling during the description of experiments)
http://chem4823.usask.ca/nmr/magnet.html
http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance
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- Practical aspects of NMR 1.instrumentation 2.Sample signal vs
water signal 3.Sample preparation (very basic aspects & deal
with specific labeling during the description of experiments)
http://www.chemistry.nmsu.edu/Instrumentat
ion/NMSU_NMR300_J.html
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- Sample preparation using recombinant methods
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- Vinarov et al., Nature Methods - 1, 149 - 153 (2004) Cell-free
protein production and labeling protocol for NMR-based structural
proteomics
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- Sample requirements and sensitivity Methyl groups are more
sensitive than isolated Ha spins Source :
www.chem.wisc.edu/~cic/nmr/Guides/Other/sensitivity-NMR.pdf
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- Sample requirements and sensitivity Cryoprobes are 3-4 times
better S/N than standard probes (2x in high salt) Source :
www.chem.wisc.edu/~cic/nmr/Guides/Other/sensitivity-NMR.pdf M not
mM!!
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- Why use NMR ? Some proteins do not crystallize (unstructured,
multidomain) crystals do not diffract well can not solve the phase
problem Functional differences in crystal vs in solution can get
information about dynamics
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- Protein Structures from an NMR Perspective Overview of Some
Basic Structural Principals: a)Primary Structure: the amino acid
sequence arranged from the amino (N) terminus to the carboxyl (C)
terminus polypeptide chain b)Secondary Structure: regular
arrangements of the backbone of the polypeptide chain without
reference to the side chain types or conformation c)Tertiary
Structure: the three-dimensional folding of the polypeptide chain
to assemble the different secondary structure elements in a
particular arrangement in space. d)Quaternary Structure: Complexes
of 2 or more polypeptide chains held together by noncovalent forces
but in precise ratios and with a precise three-dimensional
configuration.
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- Protein Structure Determination by NMR Stage ISequence specific
resonance assignment State II Conformational restraints Stage III
Calculate and refine structure
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- Resonance assignment strategies by NMR
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- Illustrations of the Relationship Between MW, c and T 2
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- NMR Assignments 3D NMR Experiments 2D 1 H- 15 N HSQC experiment
correlates backbone amide 15 N through one-bond coupling to amide 1
H in principal, each amino acid in the protein sequence will
exhibit one peak in the 1 H- 15 N HSQC spectra also contains
side-chain NH 2 s (ASN,GLN) and N H (Trp) position in HSQC depends
on local structure and sequence no peaks for proline (no NH)
Side-chain NH 2
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- 3D NMR Experiments Consider a 3D experiment as a collection of
2D experiments z-dimension is the 15 N chemical shift 1 H- 15 N
HSQC spectra is modulated to include correlation through coupling
to a another backbone atom All the 3D triple resonance experiments
are then related by the common 1 H, 15 N chemical shifts of the
HSQC spectra The backbone assignments are then obtained by piecing
together all the jigsaw puzzles pieces from the various NMR
experiments to reassemble the backbone NMR Assignments
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- 3D NMR Experiments Amide Strip 3D cube 2D plane amide strip
Strips can then be arranged in backbone sequential order to visual
confirm assignments
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- NMR Assignments 3D NMR Experiments 3D HNCO Experiment common
nomenclature letters indicate the coupled backbone atoms correlates
NH i to C i-1 (carbonyl carbon, CO or C) no peaks for proline (no
NH) Like the 2D 1 H- 15 N HSQC spectra, each amino acid should
display a single peak in the 3D HNCO experiment identifies
potential overlap in 2D 1 H- 15 N HSQC spectra, especially for
larger MW proteins most sensitive 3D triple resonsnce experiment
may observe side-chain correlations 1 J NC 1 J NH
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- NMR Assignments 3D NMR Experiments 3D HN(CA)CO Experiment
correlates NH i to C i relays the transfer through C i without
chemical shift evolution uses stronger one-bond coupling contains
only intra correlation provides a means to sequential connect NH
and C chemical shifts match NH i -CO i (HN(CA)CO with NH i -CO i-1
(HNCO) not sufficient to complete backbone assignments because of
overlap and missing information every possible correlation is not
observed need 2-3 connecting inter and intra correlations for
unambiguous assignments no peaks for proline (no NH) breaks
assignment chain but can identify residues i-1to prolines 1 J C C 1
J NH 1 J NC
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- NMR Assignments 3D NMR Experiments 3D HN(CA)CO Experiment Amide
Strips from the 3D HNCO and HN(CA)CO experiments arranged in
sequential order HNCO and HN(CA)CO pair for one residues NH
Connects HN i -CO i with HN i -CO i-1 Journal of Biomolecular NMR,
9 (1997) 1124
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- NMR Assignments 4D NMR Experiments Consider a 4D NMR experiment
as a collection of 3D NMR experiments still some ambiguities
present when correlating multiple 3D triple-resonance experiments
4D NMR experiments make definitive sequential correlations increase
in spectral resolution Overlap is unlikely loss of digital
resolution need to collect less data points for the 3D experiment
If 3D experiment took 2.5 days, then each 4D time point would be a
multiple of 2.5 days i.e. 32 complex points in A-dimension would
require an 80 day experiment loss of sensitivity an additional
transfer step is required relaxation takes place during each
transfer Get less data that is less ambiguous?
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- NMR Assignments
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- Why use deuteration? What are the advantages? What are the
disadvantages?
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- 2D 15 N-NH HSQC spectrum of the 30 kDa N-terminal domain of
Enzyme I from the E. coli Effects of Deuterium Labeling only 15 N
labeled 15 N, 2 H labeled Current Opinion in Structural Biology
1999, 9:594601
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- Protein Structure Determination by NMR Stage ISequence specific
resonance assignment State II Conformational restraints Stage III
Calculate and refine structure
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- NMR Structure Determination With The NMR Assignments and
Molecular Modeling Tools in Hand: All we need are the experimental
constraints Distance constraints between atoms is the primary
structure determination factor. Dihedral angles are also an
important structural constraint What Structural Information is
available from an NMR spectra? How is it Obtained? How is it
Interpreted?
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- 4.1 2.9 NOE CHCHCHCH NH NH CHCHCHCH J NOE - a through space
correlation (