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Great Questions and Participation in the Last Two Classes!!!!. We gave each of you maximum points!. Homework and Presentation Deadlines. Please do not delay handing in the homeworks Please do not wait until the last minute to finalize your presentation before meeting with us….

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great questions and participation in the last two classes

Great Questions and Participation in the Last Two Classes!!!!

We gave each of you maximum points!

homework and presentation deadlines
Homework and Presentation Deadlines
  • Please do not delay handing in the homeworks
  • Please do not wait until the last minute to finalize your presentation before meeting with us…

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

natively unfolded proteins and what the biophysical methods can report on them

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

Judith Klein-SeetharamanCo-Course Director

jks33@pitt.edu

lecture overview
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

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

lecture overview1
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 method for “seeing” molecules: AFM

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

natively unfolded proteins
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.

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

coil like vs pre molten globule like
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.

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

prediction of disorder
Prediction of Disorder
  • PONDR
    • Neural network from sequence features
  • SVM
  • others

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

disorder in whole genomes
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

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

disorder and function
Disorder and Function

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

lecture overview2
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 method for “seeing” molecules: AFM

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

circular dichroism tutorial
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

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

objectives of this tutorial
Objectives of this 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

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

polarized light
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

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

principle of circular dichroism
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

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

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

objectives of this tutorial1
Objectives of this 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

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

comparison of absorbance and cd
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.

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

fasman standard curves for polylysine
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

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

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

real cd spectra of example proteins
Real CD Spectra of Example Proteins

—— 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

objectives of this tutorial2
Objectives of this 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

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

obtaining secondary structure content
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

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

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

available methods
Available methods
  • Check program descriptions on package websites:

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

cdpro webinterface
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”

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

objectives of this tutorial3
Objectives of this 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

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

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

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

objectives of this tutorial4
Objectives of this 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

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

folding transitions
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.

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

folding transitions1
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.

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

links
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

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

  • Animation of polarized light

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

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

objectives of this tutorial5
Objectives of this 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

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

homework cd analysis step 1
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

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

cd data conversion
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

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

homework cd analysis step 2
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

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

datafiles for homework
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.

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

homework questions
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”?

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

how should the results look like
How should the results look like?
  • Answer the questions with the help of graphs like the ones below that you can create with the data files and the CDPro predictions

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

lecture overview3
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 method for “seeing” molecules: AFM

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

slide38

CHEMOTAXIS AND PHOTOTAXIS

Movement of cells

Movement to/away from chemicals (chemotaxis) / light (phototaxis)

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

slide39

CHEMOTAXIS AND PHOTOTAXIS

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

slide40

CHEMOTAXIS AND PHOTOTAXIS

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

slide41

CHEMOTAXIS AND PHOTOTAXIS

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

slide42

CHEMOTAXIS AND PHOTOTAXIS

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

slide43

SECONDARY STRUCTURE

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

slide44

SECONDARY STRUCTURE

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

slide45

TRANSDUCER STRUCTURE

NMR Spectroscopy

10 mM NaP pH 6.0

  • Minimal spectral dispersion
  • Negative Het-NOE values
  • Lack of NOE’s
  • Little temperature variation
  • only 1/3 of signals visible

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

slide46

TRANSDUCER STRUCTURE

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

slide47

PREDICTION OF DISORDER

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

slide48

PREDICTION OF DISORDER

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

slide49

Hypothesis

Transducer

Serine Receptor

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