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Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects

“Natively unfolded proteins” and what the biophysical methods can report on them

Judith Klein-SeetharamanCo-Course Director

jks33@pitt.edu

Lecture Overview

Natively unfolded proteins
Brief circular dichroism tutorial
Example CD: Is the transducer natively unfolded?
Brief intro to methods for “seeing” molecules
Example SANS: Is the transducer natively unfolded?
Theories on solvent effects
Alternative methods for “seeing” molecules: AFM, CryoEM outlook

Lecture Overview

Alternative method for “seeing” molecules: AFM

Natively Unfolded Proteins

X-ray crystallography defines missing electron density in many protein structures
NMR narrow chemical shift dispersion, relaxation, HetNOE, lacking NOE
CD, IR, Raman
Hydrodynamic parameters obtained from techniques such as gel-filtration, viscometry, SAXS, SANS, sedimentation, and dynamic and static light scattering
degree of globularity eg from SAXS
FRET, shape and position of the intrinsic fluorescence spectrum, fluorescence anisotropy and lifetime, accessibility of the chromophore groups to external quenchers, and steady-state and time-resolved parameters of the fluorescent dyes.
Increased proteolytic degradation
Immunochemical methods. For example, antibodies obtained against the Ca2+-saturated F1-fragment of prothrombin did not interact with the calcium-free apo-form of this protein
protein conformational stability, e.g. by calorimetric melting curve, i.e. the steepness of urea- or guanidinium chloride-induced unfolding curves depends strongly on whether a given protein has a rigid tertiary structure

Extracted from: Vladimir N. Uversky: Natively unfolded proteins: A point where biology waits for physics. Protein Science (2002), 11:739-756.

Coil-like vs. pre-molten globule like

Vladimir N. Uversky: Natively unfolded proteins: A point where biology waits for physics. Protein Science (2002), 11:739-756.

Prediction of Disorder

PONDR

Neural network from sequence features

SVM
others

Disorder in Whole Genomes

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

Disorder and Function

Lecture Overview

Circular Dichroism Tutorial

Remind you of what CD is
What data do you get typically?
How do you analyze it?
Limitations
Applications to study of dynamics and biomolecular interactions
Outline of the homework

Objectives of this Tutorial

Limitations

Polarized Light

Crystals dark

Crystals light

Linearly polarized light:

Electric vector direction constant - magnitude varies

Circular polarized light:

Electric vector direction varies - magnitude constant

staff.bath.ac.uk/bssmdb/cd_lecture.ppt

Principle of Circular Dichroism

CD measures the difference between the absorption of left and right handed circularly-polarized light. polarized light:

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

Limitations

Comparison of Absorbance and CD

Example: Native (__) versus denatured (…) DNA

Extinction coefficient at 260 nm:

De = ~3 M-1cm-1

e= ~6000 M-1cm-1

The CD signal is 0.05% of the absorbance signal.

Fasman Standard Curves for Polylysine

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

Real CD Spectra of Example Proteins

—— chymotrypsin (~all b)

—— lysozyme (mixed a & b)

—— triosephosphate isomerase (mostly a some b)

—— myoglobin (all a)

Limitations

Obtaining secondary structure content

Fit (usually using least squares minimization) the unknown curve qu to a combination of standard curves:

qt = xaqa + xbqb + xcqc

Vary xa, xb and xc

to give the best fit of qt to qu

while xa+ xb + xc = 1.0

Available methods

Check program descriptions on package websites:

CDPro Webinterface

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”

Limitations

Limitations

Strong absorption of additives (e.g. poly-ethylene-glycol, PEG, 2-Methyl-2,4-pentanediol, MPD, etc.)
Low signal to noise ratio for diluted samples
Secondary structure content not reliable, especially not for beta-sheet

Limitations

Folding Transitions

Example: Refolding of lysozyme

Transitions as a function of time after change in condition

Stopped flow CD – near UV

Stopped flow CD – far UV

Secondary and tertiary structure formation can be followed time-resolved.

Folding Transitions

Example: Transducer from Archaebacteria

Steady-state spectra as a function of change in condition

The transducer is natively unfolded under physiological conditions and becomes folded at high salt concentrations.

Links

Online and downloadable analysis tools:

Dichroweb

www.cryst.bbk.ac.uk/cdweb/html/

CDPro analysis package

http://lamar.colostate.edu/~sreeram/CDPro/

Tutorials:

Lecture similar to this one

Animation of polarized light

http://www.enzim.hu/~szia/cddemo/edemo0.htm

Limitations

Homework: CD Analysis Step 1

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

CD data conversion

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

Homework: CD Analysis Step 2

do the prediction: e.g. Continll.exe
ProtSS.out is the output file
CalcCD.out allows you to check predicted versus observed CD spectra

Datafiles for Homework

There are two sets of data, a titration in trifluoroethanol and one in ammonium sulfate.

PBS.txt (no TFE, no AS)
10TFE.txt, 20TFE.txt, 30TFE.txt, 50TFE.txt, 90TFE.txt
10as.txt, 20as.txt, 30as.txt, 50as.txt, 90as.txt

Reminder: The data is raw ellipticity data, it needs to be converted: [Q]mrw = [Q]/(10*l*c*n) where [Q] is the raw ellipticity, l is the cell path-length in cm, c is the protein concentration in M, n is the number of peptide bonds in the protein. This data is from pHtrII-cyt (28800 Da) that was measured at 0.1 mg/ml in a 0.2 cm cell.

Homework Questions

Predict secondary structure content for the two datasets (ammonium sulfate titration, trifluoroethanol titration)
For each prediction, view CalcCD.out to check predicted versus observed CD spectra
Compare the predictions by two different methods (Continll.exe etc.) (ProtSS.out is the output file)
Compare for at least one dataset and one method the use of different reference protein datasets
Rationalize what reference dataset makes sense to use in this case
Are there any differences between the two different “folding agents”?