Structure and Behaviour of Proteins, Nucleic Acids and Viruses from Raman Optical Activity Laurence Barron

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Structure and Behaviour of Proteins, Nucleic
Acids and Viruses from Raman Optical
ActivityLaurence Barron

• Ewan Blanch
• Iain McColl
• Lutz Hecht
• Supported by EPSRC and BBSRC

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Chirality
I call any geometrical figure or group of points
chiral, and say that it has chirality if its
image in a plane mirror, ideally realized, cannot
be brought into coincidence with itself. Lord
Kelvin, Baltimore Lectures, 1884
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Raman Spectroscopy

• Provides vibrational spectra via
• inelastic scattering of visible light.
• Analyze visible scattered light with
• a visible spectrograph.
• Multichannel detection gives
• full vibrational spectrum from
• 50-4000 cm-1 in a single
• acquisition.

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Raman Optical Activity (ROA)

• We measure ROA as a tiny difference in the
  intensity of Raman scattering from chiral
  molecules in right (R)- and left (L)-circularly
  polarized incident light

• ROA (and VCD) provides vibrational optical
  activity spectra. Like visible and
  ultraviolet CD, it is sensitive to chirality, but
  via vibrational rather than electronic
  transitions.

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First Observations of ROA
L. D. Barron, M. P. Bogaard and A. D. Buckingham.
J. Am. Chem. Soc. 95, 603 (1973)
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Progress!
(R)-()
1972
2003
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ROA of Microsamples

• Microgram quantities held
• in capillary tubes!
• Recorded on the Zurich prototype
• of the new commercial ROA
• instrument from BioTools, Inc.
• W. Hug, Appl. Spectrosc. 57, 1 (2003).

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L. D. Barron and A. D. Buckingham, Mol. Phys. 20,
1111 (1971)
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Two-Group Model of ROA

• ROA is generated by interference between light
  waves scattered independently from two achiral
  anisotropic groups held in a twisted chiral
  arrangement. L. D. Barron and A. D. Buckingham,
  J. Am. Chem. Soc. 96, 4769 (1974).
• Predicts zero ROA in forward scattering and
  maximum ROA in backscattering.
• Backscattering is essential for ROA measurements
  on biomolecules in aqueous solution because
  fluctuations from the high water and fluorescence
  background tends to swamp the ROA signals.

wavenumber
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The Glasgow Backscattering ROA Instrument
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Analysis of ROA Spectra

• ROA spectra of small chiral molecules are best
  analyzed using ab initio computations (P. L.
  Polavarapu). Absolute configuration can be
  assigned from an ab initio calculation which
  correctly predicts the signs of most observed ROA
  bands.
  CHFClBr J. Costante, L.
  Hecht, P. L. Polavarapu, A. Collet and L. D.
  Barron. Ang. Chem. Int. Ed. Engl. 36, 885 (1997).
• For large biomolecules like proteins,
  conformational elements are identified from a
  comparison of ROA band patterns with patterns
  seen in molecules of known structure (from X-ray
  crystallography or NMR).

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Biomolecular Structure

• ROA is more incisive than conventional Raman
  spectroscopy in the study
• of biomolecules. Vibrations which sample the
  skeletal chirality most directly make the largest
  contributions to the ROA intensity.
• Conventional Raman spectra of proteins are
  dominated by side chain bands which tend to
  obscure the backbone bands. But ROA spectra are
  dominated by peptide backbone bands and so give
  more direct information about secondary and
  tertiary structure.
• ROA reaches those parts other spectroscopies
  fail to reach!

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Hen Lysozyme
a
a
a b
Trp
backbone skeletal stretch
a b
b
side chains
amide I
extended amide III
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Human Immunoglobulin
b-turn
PPII
b
b-turn
b
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Protein SuperfoldsNine families of
protein superfold structures are found to
represent 46 of all non-homologous (i.e. having
neither sequence nor functional similarity)
proteins in the protein data bank. (J. M.
Thornton, 1995)
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Pattern Recognition in Protein ROA Spectra

• Protein fold information obtained from ROA
  spectra using pattern recognition methods such as
  principal component analysis (PCA).
• From the set of experimental ROA spectra, PCA
  calculates a set of sub-spectra, the algebraic
  combination of which with appropriate
  coefficients can be used to reconstruct any
  member of the original set of ROA spectra.
• A scatter plot of the two most important
  coefficients against each other reveals
  structural relationships among the members of the
  set. The proteins cluster together according to
  structural type.
• Collaborator Kurt Nielsen, Technical University
  of Denmark.

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Glycoprotein ROA

• Polypeptide and carbohydrate bands
• visible.
• Orosomucoid (a1-glycoprotein) clearly
• mainly b-sheet (lipocalin up-and-down
• b-barrel?).

b-lactoglobulin

• Invertase appears to have no secondary
• structure. Most bands are carbohydrate.

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Protein Misfolding and Disease
Alzheimers, Parkinsons, the prion
encephalopathies (scrapie, BSE, CJD), and
amyloidosis are protein misfolding diseases
involving formation and tissue deposition of
amyloid fibrils.
unfolded protein

partially folded intermediate
extended cross b-sheet formation
folded protein
amyloid fibrils (transthyretin) C. Blake and L.
Serpell Structure 4, 989 (1996)
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Polypeptides in Model Conformations
poly(L-glutamic acid)
poly(L-lysine)
pH 4.8, 200C R (CH2)2CO2H a-helix
pH 11, 30C R (CH2)4NH2 a-helix
pH 3, 200C R (CH2)4NH3 disordered
pH 12.6, 200C R (CH2)2CO2- disordered
pH 11, 500C R (CH2)4NH2 b-sheet
b-sheet poly(L-glutamic acid) ?

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Poly(L-Proline) II Helix and Disordered
Polypeptides

• From UVCD data, Tiffany and Krimm (1968)
  suggested PPII helix is the main
• conformational element in disordered
  poly(L-lysine) and poly(L-glutamic acid).
• Reinforced from VCD data by Dukor and Keiderling
  (1991)
• and critically reviewed by Woody (1992).
• PPII extensively discussed in Adv. Prot. Chem.
  62 (2002).

Left-handed f -780, y 1460. Threefold
rotational symmetry (n 3). No intrachain
hydrogen bonds. Stabilized by hydrogen bonding to
water (backbone and sidechain).

• The plastic, adaptable character of PPII has
  functional importance
• (molecular recognition, binding etc.).

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ROA of Poly(L-Proline) II Helix
Take ROA of disordered poly(L-glutamic acid) as
characteristic of PPII helix? OOAAAAAAAOO
peptide shown by NMR and CD to be mainly
PPII helix in aqueous solution. Shi et al. PNAS
99, 9190 (2002).
OOAAAAAAAOO
ROA
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The Amyloidogenic Prefibrillar Intermediate of
Human Lysozyme

• Although most proteins simply form amorphous
  aggregates
• under denaturing conditions, human lysozyme
  forms amyloid fibrils if incubated at 56 0C at pH
  2.0.
• The prefibrillar intermediate is molten
  globule-like, and it survives as a monomer for up
  to 24 hours, long enough for ROA measurements.
• Collaborator Ludmilla Morozova-Roche, Oxford/Umea

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Native and Prefibrillar Human Lysozyme

• Hydrated a-helix band has disappeared.
• Tryptophan W3 band has disappeared.
• PPII band has appeared.

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Poly(L-Proline) II Helix

• Left-handed.
• No intrachain
• hydrogen bonds.
• Stabilized by
• hydrogen bonding
• to water.

• Elimination of water molecules
• between PPII strands to form b-sheet
• is highly favourable entropically.
• Is PPII helix the killer conformation?
• E. W. Blanch et al. J. Mol. Biol.
• 301, 553 (2000).
• Adv. Prot. Sci., 62 (2002).

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a-Synuclein

• Abundant in brain tissue but function
• unknown. Highly fibrillogenic.
• Aggregation into amyloid fibrils is
• associated with Parkinsons disease.
• Unfolded in its native state. ROA shows it
• to contain mostly PPII structure.
• b- and g-synuclein are not fibrillogenic
• but ROA shows they have similar
• structures. Must also consider residue
• properties like charge and hydrophobicity.
• Collaborator Michel Goedert,
• MRC LMB, Cambridge.
• C. D. Syme et al. Eur. J. Biochem.
• 269, 148 (2002).

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The Prion Protein

• In prion disease, the normal cellular form
• of the prion protein PrPC converts to a
• scrapie amyloid form PrPSc.
• Solution NMR structure of the normal
• form reveals a folded C-terminal domain
• plus a disordered N-terminal tail.
• ROA reveals disordered tail is mostly PPII.
• Collaborator Andrew Gill, Institute for
• Animal Health, Compton.

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A-type RNA Double Helix
C3-endo
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ROA of Natural DNA and RNA Molecules
C3-endo
G,A
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Molecular Structures of Viruses

• Knowledge of virus structure at the molecular
  level is essential for understanding how they
  work, but for most nothing is known about the
  protein and nucleic structures.
• Most viruses cannot be studied using X-ray
  diffraction or NMR. The few dozen X-ray
  structures, crystal or fibre, are immensely
  valuable, but usually only the coat proteins are
  seen, the nucleic acid core being too disordered.
  
• ROA provide information about both protein and
  nucleic acid structures.

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Filamentous Bacteriophages
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Tryptophan Absolute Stereochemistry
1050
T. Miura, H. Takeuchi and I Harada, J. Raman
Spectrosc. 20, 667 (1989). E.W. Blanch et al.,
J. Am. Chem. Soc. 123, 4863 (2001).
-930
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Tobacco Mosaic Virus (TMV)
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Satellite Tobacco Mosaic Virus (STMV)
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Cowpea Mosaic Virus (CPMV)

• Type member of the comovirus group of
• plant viruses.
• Preparations separate into three bands by
• centrifugation on CsCl density gradient.
• Bipartite genome of separately encapsidated
• RNA-1 and RNA-2 molecules.

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CPMV Nucleic Acid
RNA-2

• Subtraction of the ROA spectrum of
• the top component (empty protein
• capsid) from those of the middle and
• bottom components provides ROA
• spectra of the RNA-1 and RNA-2
• cores.
• The ROA of RNA-1 and RNA-2 are
• almost identical.
• Very similar to ROA of tRNAPhe.
• A-type single-stranded helix.

RNA-1
tRNA(Phe)
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• Summary
• The same basic ROA instrument gives high quality
  spectra of a vast range of chiral structures,
  from the smallest such as CHFClBr to the largest
  such as intact viruses.
• The sensitivity of ROA to chirality makes it an
  incisive probe of the structure and behaviour of
  the molecules of life.
• A commercial ROA instrument is now available from
  BioTools (www.btools.com info_at_btools.com).