Application of NMR techniques to biomacromolecules

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Application of NMR techniques to
biomacromolecules

• Structural study (proteins secondary, tertiary
  structures)
• Assignment of proton, intra-residue interaction
  by correlation spectroscopy13C and 15N (31P for
  oligonucleotides) resonances using NOE
  relationship to determine the proton-proton
  distances (lt5 Å) scalar coupling constant
  (dihedral angle) using primary sequence,
  pattern recognition, distance geometry and
  simulated annealing or restraint molecular
  dynamics to obtain convergence in structure.

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Application of NMR techniques to
biomacromolecules

• Dynamics study from relaxation rate enhancement
  to deduce exchange rate and diffusion
  coefficient protein folding using
  proton/deuterium exchange measurements and
  stopped-flow, and quenching, photo-activation
• Chemical exchange and binding studytransferred
  NOE relaxation enhancement by paramagnetic
  species (e.g. Mn2) or spin labels

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References
1.    A. Abragam, Principles of Nuclear
Magnetism, Clarendon Press, Oxford, 1961. 2.   
K. Wüthrich, NMR of Proteins and Nucleic acids,
John Wiley Sons, N.Y., 1986. 3.    I. Solomon,
Phys. Rev. 99, 559-565, 1955. 4.    J. Cavanagh,
W.J. Fairbrother, A.G. Palmer III and N.J.
Skelton, Protein NMR Spectroscopy Principles and
Practice, Academic Press, San Diego,
1996.  5.   J.N.S. Evans, Biomoleculae NMR
Spectroscopy, Oxford University Press, Oxford,
1995.  
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Dynamics Spectral density function
J(w)2/5tc/(1 w2tc2) wheretc is the
correlation time which is a function of
temperature, solvent viscosity and molecular
size.
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T1-1 (3/2)g4?2I(I1)J(1)(wI)J(2)( 2wI) T2-1
g4?2I(I1)3/8J(0)(0)15/4J(1)( wI)3/8 J(2)(
2wI) Longitudinal and transverse relaxation by
Scalar or J coupling between interacting nuclei
(spin number I) Spin-spin coupling between a pair
of nuclei via electrons in the bonds between them
is the mechanism for J coupling. This results in
a splitting of the resonance. It is an important
basis of correlation spectroscopy.
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Chemical exchange for a two spin system Chemical exchange for a two spin system
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Short distances found in protein structures
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Typical ranges of scalar coupling constants
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Dynamic characteristics of protein structure
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The strategy for solving three-dimensional
structure of biological macromolecules on the
basis of NMR data
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Transmission IR
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References

C.R. Cantor and P.R. Schimmel, Biophysical
Chemistry, W.H. Freeman and Co., New York, 1980.
R.F. Steiner and L. Garone, The Physical
Chemistry of Biopolymer Solutions, World
Scientific Co. , Singapore, 1991.
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Surface Plasma Resonance
refractive index (RI) as a function of medium
density analyte immobilized on e.g. dextran
which is bound to a gold surface binding of
substrate to analyte results in RI change can be
used in kinetics and binding studies
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Surface plasmon resonance
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Glass
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T20 interation with gp41 N-terminal
peptide gp41(516-566)
Ka 5.66e6 Kd1.77e-7
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Circular dichroismto measure the secondary
structures of proteins
Linearly polarized light can be decomposed into
right- and left-hand (RH and LH) circularly
polarized light when the polarized light is
absorbed by optically active compounds, there may
be a difference in the absorbance between RH and
LH polarized lights, and thus circular dichroism.
Proteins with regular secondary structures
exhibit distinct pattern of CD spectra which ca
be used to deduce the secondary structures of the
proteins.
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Effect of an optically active absorbing sample on
incident linearly polarized light. All drawings
show the electric field vector viewed along the
direction of light propagation. Points 1 through
5 correspond to equal (increasing) time
intervals. (a) Incident linearly polarized
light. (b) Elliptically polarized light produced
by passing the incident light through an
optically active sample. (c) Resolution of
linearly polarized light into individual
right-hand and left-hand circularly polarized
components. (d) Effect of an optically active
sample on the two circularly polarized
components. The sum of measurements made with
these two separate components must be identical
to the result obtained in part b.
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Circular Dichroism Spectroscopy
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References

J.R. Lakowicz, Principles of Fluorescence
Spectroscopy. Plenum Press, New York, 1999.
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Fluorescence spectroscopy

• Membrane binding studyemission wavelength and
  intensity change (NBD) fluorescence
  quenchingTrp and acrylamide
• Self-assembly of biomacromoleculesself-quenching
  of fluorophore, e.g. rhodamine
• Membrane fusionfluorescence resonance energy
  transfer (FRET) between, e.g. NBD and
  Rhodamine-labeled phospholipids

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Fluorophores
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Pathways for production and deexcitation of an
excited state
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Spectral overlap for fluorescence resonance
energy transfer (RET)
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Fluorescence energy transfers dependence on gp41
fusion peptide acceptor concentration