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

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 • 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 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 • PONDR • Neural network from sequence features • SVM • others Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects

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

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 • 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 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 • 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

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 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 —— 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

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 • Check program descriptions on package websites: Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects

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

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

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 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 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

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 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 • 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 • 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 • 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? • 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

CHEMOTAXIS AND PHOTOTAXIS Movement of cells Movement to/away from chemicals (chemotaxis) / light (phototaxis) Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects

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

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

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

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

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

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

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

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

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

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

Hypothesis Transducer Serine Receptor Molecular Biophysics 3: Lecture “Natively Unfolded Proteins” and Solvent Effects

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