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Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Judith Klein-SeetharamanCo-Course Director
jks33@pitt.edu
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Extracted from: Vladimir N. Uversky: Natively unfolded proteins: A point where biology waits for physics. Protein Science (2002), 11:739-756.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Vladimir N. Uversky: Natively unfolded proteins: A point where biology waits for physics. Protein Science (2002), 11:739-756.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Prevalent Structural Disorder in E. coli and S. cerevisiae Proteomes: Peter Tompa,* Zsuzsanna Doszt nyi, and Istv n Simon J. Proteome Res., 5 (8), 1996 -2000, 2006.
Prediction and Functional Analysis of Native Disorder in Proteins from the Three Kingdoms of Life: J. J. Ward , J. S. Sodhi , L. J. McGuffin , B. F. Buxton and D. T. Jones Journal of Molecular Biology 337, Issue 3 , Pages 635-645
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Crystals dark
Crystals light
Electric vector direction constant - magnitude varies
Electric vector direction varies - magnitude constant
staff.bath.ac.uk/bssmdb/cd_lecture.ppt
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
http://www.cryst.bbk.ac.uk/BBS/whatis/cd_website.html
DA(l) = AR(l)-AL(l) = [eR(l) - eL(l)]lc
or
DA(l) = De (l)lc
staff.bath.ac.uk/bssmdb/cd_lecture.ppt
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Example: Native (__) versus denatured (…) DNA
De = ~3 M-1cm-1
e= ~6000 M-1cm-1
The CD signal is 0.05% of the absorbance signal.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
80000
EL – ER > 0
EL – ER < 0
60000
a
-helix
b
-sheet
40000
random coil
20000
Mean residue ellipicity in deg cm2dmol-1
0
-20000
-40000
190
200
210
220
230
240
250
wavelength in nm
staff.bath.ac.uk/bssmdb/cd_lecture.ppt
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
—— chymotrypsin (~all b)
—— lysozyme (mixed a & b)
—— triosephosphate isomerase (mostly a some b)
—— myoglobin (all a)
staff.bath.ac.uk/bssmdb/cd_lecture.ppt
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
qt = xaqa + xbqb + xcqc
to give the best fit of qt to qu
while xa+ xb + xc = 1.0
staff.bath.ac.uk/bssmdb/cd_lecture.ppt
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
If you want to run the program within the webbrowser click ReadMe
If you want to download the program to a PC, click “CDPro.zip”
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Example: Refolding of lysozyme
Stopped flow CD – near UV
Stopped flow CD – far UV
Secondary and tertiary structure formation can be followed time-resolved.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Example: Transducer from Archaebacteria
The transducer is natively unfolded under physiological conditions and becomes folded at high salt concentrations.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Online and downloadable analysis tools:
www.cryst.bbk.ac.uk/cdweb/html/
http://lamar.colostate.edu/~sreeram/CDPro/
Tutorials:
staff.bath.ac.uk/bssmdb/cd_lecture.ppt
http://www.enzim.hu/~szia/cddemo/edemo0.htm
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Use the CDPro package to analyze primary CD data of protein X. http://lamar.colostate.edu/~sreeram/CDPro/main.html
Original data:
Convert the two raw data files into files that are readable for the CDpro program by using CRDATA.exe
Column 1: wavelength (should start from longer wavelength, e.g. 200 nm to 100 nm)
Column 2: mean residue ellipticity (not molar ellipticity, teta)
Separated by tab
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Mean residue ellipticity (ΘMRW) and molar ellipticity (Θ) are related as follows:
where
l=pathlength in cm, typically 0.2 cm
c=concentration in M,
n=number of peptide bonds in protein,
Θ=raw ellipticity in mdeg
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Movement of cells
Movement to/away from chemicals (chemotaxis) / light (phototaxis)
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
The Receptors
Ligand
Conserved properties:
Functions as dimer
HAMP domain
Methyl-accepting signaling protein MCP domain
Light
Examples: Tar, Tsr from E. coli
Transducer HtrII from N. pharaonis
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Activation Mechanism Models
Mechanical models
“Dynamic” models
Ottemann K.M., Science, 285 (1999), pp. 1751-1754
Kim S.-H., Prot.Sci., 3 (1994), pp. 159-165
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Structures
Ligand
Light
Piston or Rotation
Rotation/
Displace-ment
Changes in Dynamics
?
Nothing is known about the cytoplasmic domain of the phototaxis transducer
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Goal
Secondary Structure Prediction:
Tertiary Structure Prediction:
Investigate the structure and dynamics of the cytoplasmic domain of the phototaxis transducer of N. pharaonis
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Circular dichroism in PBS
HtrII-cyt is a random coil? That would make it an intrinsically unstructured protein.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Circular dichroism with additives
PBS
4M KCl
Ammonium sulfate
Far-UV CD spectra of pHtrII-cyt in PBS (solid line), PBS plus 4 M KCl
(dotted line), PBS plus 40% ammonium sulfate (dashed-dotted line), and the
dimer peak of pHtrII-cyt after cross-linking in PBS plus 4MKCl in PBS (dashed
line).10
Under native conditions (4M KCl), 19% helix is detected.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
NMR Spectroscopy
10 mM NaP pH 6.0
10 9 8 7 6 5 4 3 2 1 0
125 120 115 110
15N chemical shift, ppm
8.5 8.0 7.5 7.0 6.5
1H chemical shift, ppm
Highly dynamic with evidence for intermediate conformational exchange.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
FTIR
10 mM Tris-HCl pH 9.0 in D2O
Dry film
1654
1644
adapted from Stuart B. (1997), Biological Applications of Infrared Spectroscopy, University of Greenwich, UK
Dehdyration induces helix.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Using PONDR
The transducer cytoplasmic domain is predicted to be more disordered than the serine and aspartate receptors.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Quantitative comparison of disorder in MCP family
Maximum at each position in the alignment
Transducer
Mean
Serine Receptor
Minimum at each position in the alignment
Disorder in serine receptor follows the mean, in transducer is significantly above the mean.
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects
Transducer
Serine Receptor
Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects