CD and Fluorescence

1
CD and Fluorescence

• Overview of the theory, technique and its
  applications

2
Overall

• Three take home messages
• What is the theory behind the technique?
• What is the basic technique?
• What are the applications?
• Real life examples
• What are the question(s)?
• How they used the techniques?
• What were the answers?

3
Plane-polarized waves

• If the vector of the electric field (measured at
  a fixed point of space) oscillates along a
  straight line then the waves are called
• plane-polarized or linearly polarized waves
• The following animation presents a wave that is
  plane-polarized in a vertical plane.

(Attention! The two animations are not
synchronized!)
4
Plane-polarized waves

• Same thing horizontal plane
• If the vector of the electric field (measured at
  a fixed point of space) oscillates along a
  straight line then the waves are called
• plane-polarized or linearly polarized waves
• The following animation presents a wave that is
  plane-polarized in a vertical plane.

(Attention! The two animations are not
synchronized!)
5
Circular polarized waves

• Superposition of two waves that have the same
  amplitude and wavelength and are polarized in two
  perpendicular planes but there is a phase
  difference of 90 between them
• A phase difference of 90 means that when one
  wave is at its peak then the other one is just
  crossing the zero line.

(Attention! The two animations are not
synchronized!)
6
Circular polarized waves

• At any fixed point in space that is in the line
  of the propagation of this wave, the electric
  field vector rotates in a circle while its length
  remains constant.
• Such waves are called circularly polarized
  waves.

(Attention! The two animations are not
synchronized!)
7
Circular polarized waves

• Same thing different direction
• At any fixed point in space that is in the line
  of the propagation of this wave, the electric
  field vector rotates in a circle while its length
  remains constant.
• Such waves are called circularly polarized
  waves.

(Attention! The two animations are not
synchronized!)
8
Circular Dichorism

• Clockwise

• Counter-clockwise

(Attention! The four animations are not
synchronized!)
9
Superposition of circularly polarized waves

• Any linearly polarized light wave can be obtained
  as a superposition of a left circularly polarized
  and a right circularly polarized light wave,
  whose amplitude is identical.

red and green colors indicate the two superposing
components and light blue indicates the resulting
wave
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The interaction of light and matter

• If light enters matter, its properties may
  change.
• Namely, its intensity (amplitude), polarization,
  velocity, wavelength, etc. may alter.
• The two basic phenomena of the interaction of
  light and matter are absorption (or extinction)
  and a decrease in velocity.

11
Plane-polarized waves in a medium showing
circular dichroism
12
Plane-polarized waves in a medium showing
circular dichroism

• Any linearly polarized light can be obtained as
  the superposition of a left circularly polarized
  and a right circularly polarized light wave.

• Some materials possess a special property they
  absorb left circularly polarized light to a
  different extent than right circularly polarized
  light.
• This phenomenon is called circular dichroism.

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Circular Dichroism

• Theory of the technique
• Optically active matter (proteins/DNA/RNA/
• carbohydrates/etc) absorbs left and right hand
  circular polarized light differently

14
Circular Dichroism

• Far UV-CD of random coil
• Positive at 212 nm (pgtp)
• Negative at 195 nm (ngtp)
• Far UV-CD of ß-sheet
• Negative at 218 nm (pgtp)
• Positive at 196 nm (ngtp)
• Far UV-CD of a-helix
• Coupling of the pgtp transitions leads to
  positive (pgtp) perpendicular at 192 nm and
  negative (pgtp) parallel at 209 nm
• Negative at 222 nm is red shifted (ngtp)

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Circular Dichroism

• Basics of the technique
• Small sample volumes
• 200mL
• 5-30 minute data collection
• Short data collection time
• 5-30 minute analysis time
• Short analysis time
• Recovery of sample

• Near UV spectrum
• Probe changes in tertiary structures
• No standards available
• Every protein is slightly different
• Probe overall global changes

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Circular Dichroism

• Applications
• CD has an important role in the structural
  determinants of proteins or DNA or RNA, etc
• The real power of CD is in the analysis of
  structural changes in a protein or DNA or RNA
  upon some perturbation, or in comparison of the
  structure of an engineered protein or DNA or RNA
  to the parent protein or DNA or RNA.
• CD is rapid and can be used to analyze a number
  of candidate proteins from which interesting
  candidates can be selected for more detailed
  structural analysis like NMR or X-ray
  crystallography.

17
Conformational Changes

• Here is our system
• Calbindin D28k
• Involved in apoptosis regulationwhich is highly
  expressed in brain tissue
• Question they are asking
• Can a Ca2 binding protein bind Zn2?

18
CD Results

• Far UV Near UV
• 2o structure 3o structure

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Conclusion

• What they found out
• Zn2-bound state is structurally distinct from
  the Ca2-bound state

20
Just one

• That is just one example
• There are many things you can do with CD
• kinetic measurements, melting curves

21
Fluorescence

• Theory of the technique
• Excitation at a specific wavelength
• Excitation of a fluorophore at three different
  wavelengths
• (EX 1, EX 2, EX 3) does not change the emission
  profile
• but does produce variations in fluorescence
  emission intensity (EM 1, EM 2, EM 3) that
  correspond to the amplitude of the excitation
  spectrum.

22
Fluorescence

• Basics of the technique
• Very simple
• Light source excites sample
• Emission is detected and record
• What is difficult?
• Choice of probes

23
Fluorescence

• Basics of the technique
• Small sample volumes
• 200mL 1mL
• 5-30 minute data collection
• Short data collection time
• 5-30 minute analysis time
• Short analysis time
• Recovery of sample
• Menable to many different types of expt setup
• Kinetic expts, titration expts, etc

24
Intrinsic Fluorescence

• Tryptophan, the dominant intrinsic fluorophore,
  is generally present at about 1mol in proteins.
• Trp is very sensitive to its local environment.
• It is possible to see changes in emission spectra
  in response to
• Conformational changes, subunit association,
  substrate binding, denaturation, and anything
  that affects the local environment surrounding
  the indole ring.
• Also, Trp appears to be uniquely sensitive to
  quenching, either by externally added quenchers,
  or by nearby groups in the protein.
• Trp fluorescence can be selectively excited at
  295-305 nm. (to avoid excitation of Tyr)

25
Fluorescence

• Applications
• Fluorescence has an important role in the
  structural determinants of proteins, DNA or RNA,
  etc
• Binding studies
• Structural perturbation upon binding
• Unfolding studies
• Determine intermediate states of folding
• Orientation of molecules
• Binding occurs on the N-terminal or C-terminal
• Analysis of hydrophobic surfaces
• Upon binding ligand, hydrophobic surfaces exposed
  to bind another molecule

26
Conformational Changes

• Here is our system
• Historically two types of Ca binding proteins
• Buffers
• Bind extra-cellular Ca
• Calbindin D9K
• Sensors
• Bind Ca then bind targets
• Calmodulin

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Extrinsic Probes
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Extrinsic Probes

• ANS fluorescence
• 385 nm excitation
• 400 600 nm scan
• Calbindin D9K standard
• Ca2 buffer no conformational change
• Calmodulin standard
• Ca2 sensor conformational change
• Calbindin
• This is our known

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Extrinsic Probes

• Effect of pH on the ANS binding to Ca2 -free and
  Ca2 - loaded calbindin D28k.
• Fluorescence spectra of 60 µM ANS in the presence
  of 12 µ M human recombinant calbindin D28k.
• Probe structural changes due to pH changes

30
Just one

• Fluorescence Resonance Energy Transfer
• A donor chromophore in its excited state can
  transfer energy by a nonradiative, long-range
  dipole-dipole coupling mechanism to an acceptor
  chromophore in close proximity (typically lt10nm).