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MOAC Doctoral Training CentreNovember/December 2008

CH921:Data Acquisition I

Biophysical Techniques

Course Information

Course leader: Dr. Ann Dixon Contributors: Prof. Alison Rodger; Dr. Claudia Blindauer;

Dr. Steven Brown; Dr. Vilmos Fulop, Dr. Sue Slade

CH921: Biophysical Techniques Table of Contents Nov/Dec 2008

TABLE OF CONTENTS. Page

Deadlines and other important information 3Timetable 4Essay assignment 5Laboratory and workshop manual: 6

Aims and Assessment 7-8Basic Laboratory Skills 9-10Introduction to Protein Databases Workshop 11-12Dichroweb CD Data Fitting Workshop 13-15Experiment I 16-17Experiment II 18Experiment III 19-23Experiment IV 24Experiment V 25DNA Melting Curve Exercise 26-27

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CH921: Biophysical Techniques Deadlines Nov/Dec 2008

The following contains important information regarding deadlines, submission of work, etc. Please read CAREFULLY.

Attendance and Submission of Work: Attendance at all scheduled sessions will be mandatory and recorded. Please ensure Mrs. Monica Lucena has your correct email address. You will be notified of

timetable changes by email with at least 24 hours notice. Failure to note timetable changes will result in loss of credit for attendance.

IAMBEC students to submit all work to Mrs. Christina Forbes. MOAC students to submit all work to Mrs. Monica Lucena. Plagiarism policy: Any text directly cut and pasted from the internet or any online or

electronic source will be automatically regarded as plagiarism. In cases where a particular phrase is reproduced directly from a published source (of any type), then the source should be referenced in full at the point at which it is quoted. Furthermore, the amount of directly reproduced phrases should be minimal and limited to what is essential to support the arguments presented in the text. In any case the total amount of directly reproduced (and referenced) phrases should not exceed 5% of the full piece of work. Complex diagrams, which would otherwise be difficult to reproduce, may be taken from a published source provided that the source is directly referenced and the appropriate reproduction permission has been achieved, if required (not needed for essays or laboratory reports).

Assessed work and Deadlines:Deadlines are serious. 1% / hour late; 3%/day late unless written extension from Dr. A. Dixon.

Workshop problems/proof of completion: Due by 5 pm on the day of the workshop. Laboratory reports: Due Monday 8th December by 12 noon. Essay: Due Tuesday 25th November at 12 noon. DNA melting exercise: Due Tuesday 25th November at 12 noon. NMR assessment: Due Tuesday 2nd December at 12 noon.

An oral examination (with 2 hours minutes written/reading work before hand, to be submitted at the oral) will take place on Monday 1st December. You may take up to one A4 sheet of handwritten notes only into the written part of the examinations.

Breakdown of Marks: Assessed work: 45% Exam: 45% Attendance: 5% Laboratory conduct: 5%

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CH921: Biophysical Techniques Timetable Nov/Dec 2008

CH921 TIMETABLE. Timetable includes times, dates, academic lecturer, and location

Week 6: 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00Mon. Nov. 3 UV workshop

Rodger MOAC Fluorescence lecture

& workshop (DNA melting curve)

Rodger MOAC

CD lecture Rodger MOAC

Dichroweb workshop Rodger

Tues. Nov. 4 Mass Spectrometry Slade MOAC

Pre-labMOAC(12:30)

Laboratory: Experiments I & II Dixon Chemistry B309

Week 7: 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00Mon. Nov. 10 Linear Dichroism

Rodger MOACDatabases workshop

Dixon MOACTues. Nov. 11 Pre-lab

MOACLaboratory: Exp. III

Dixon Chemistry B309Week 8: 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00

Mon. Nov. 17 Introduction to NMR Brown MOAC

NMR Group Work Brown MOAC

Tues. Nov. 18 Pre-lab MOAC

Laboratory: Exp. IV Dixon Chem B309

Solid State NMR Tutorial (Brown): Group 1: 10:00-11:00 am, Group 2: 11:15-12:15 amSolution State NMR Tutorial (Prokes):

Week 9: 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00Mon. Nov. 24 Bio-applications of high field NMR

Blindauer MOACX-Ray crystallography

Fulop MOACTues. Nov. 25 Deadline: Essay and DNA melting

exercise due (12:00)12:30

Pre-labMOAC

Laboratory: Exp. VDixon Chem B309

Week 10: 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00Mon. Dec. 1 EXAMTues. Dec. 2 Deadline: NMR Assessment due

(12:00)Crystallography demo/practical

Fulop Biol. Sci.Week 10+1: 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00

Mon. Dec. 8 Deadline: Lab reports & FEEDBACK FORM due (12:00)

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CH921: Biophysical Techniques Essay Nov/Dec 2008

ESSAY. Discuss the discovery of the structure and function of monoclonal antibodies with reference to a particular protein of your choice. Write your essay from the viewpoint of the biophysical techniques used to characterize them. Include in your essay (this means it must be a structured piece of text that hangs together not isolated notes on different techniques – it must tell a ‘story’) consideration of how NMR, absorbance spectroscopies, X-ray crystallography and mass spectrometry were/might have been used.

Write using an American Chemical Society Journal template (such as Biochemistry, see below for appropriate web sites).

Use at least 3 primary literature references.

Write 15002000 words plus any diagrams (Figure captions do not count in word count). Make sure all tables and figures are self contained and also make sure all tables and figures are referred to in the text.

Marks will be given for content and also spelling, grammar, setting out etc.

Both an electronic and hard copy version should be submitted.

Web sites to lead you to the Biochemistry template and instructionshttp://pubs.acs.org/http://pubs.acs.org/about.htmlhttp://pubs.acs.org/journals/bichaw/index.htmlhttps://paragon.acs.org/paragon/application?pageid=content&parentid=authorchecklist&mid=ag_bi.html&headername=Author%20Guidelines%20-%20Biochemistryhttps://paragon.acs.org/paragon/application?pageid=content&parentid=authorchecklist&mid=mt_bi.html&headername=Biochemistry%20Templates

All references you use should be relatively recent.

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CH921: Biophysical Techniques Laboratory and Workshop Manual Nov/Dec 2008

____________________________________

MOAC Doctoral Training Centre

CH921Data Acquisition I

Biophysical Techniques

Laboratory and Workshop Manual

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CH921: Biophysical Techniques Introduction and Assessment Nov/Dec 2008

INTRODUCTION. The aims of this lab course are to familiarize you with biological samples and teach you competence in a set of standard techniques. These include:

COSHH and solution preparation (Basic Lab Skills and Experiment I).

Protein and DNA sample preparation (Experiment II).

Protein concentration determination (Experiment III).

Protein secondary structure determination by circular dichroism (Experiment IV).

Equilibrium binding constant determination by fluorescence spectroscopy: DNA and ruthenium tris(1,10-phenanthroline) (Experiment V).

* You will be working in pairs in lab, but you will be responsible for independently writing up your lab reports.

** Important: Please read the relevant lab scripts BEFORE coming to lab - you must be prepared in order to complete experiments in the allocated time.

ASSESSMENT. Hand in a brief description of the experiments you performed (enough detail so you could look up your notes during your project and use the techniques), plots of the spectra you have recorded and the structural deductions you can make from them. Demonstrators will also be giving you a grade for laboratory work. Aspects being assessed will include:

o Improvement of laboratory skills with respect to sample handling

o Tidiness and cleanliness

o Accuracy of results

o Care of equipment

o Organisation and efficiency in the laboratory.

Your laboratory reports may form the basis of part of your oral examination for this module. Also include information from the databases about the protein you have worked with including: molecular weight, amino acid residue content, extinction coefficient, -helix content as determined from the crystal structure.

BEFORE COMING TO LAB:

Read lab scripts for the current day.

Perform all required calculations - lab time is limited and you will not be allowed to stay longer than scheduled session.

Obtain lab coat, safety glasses and lab book and bring ALL THREE to every lab session. If you do not have all of these items as well as your lab manual, you will be asked to go get these items.

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CH921: Biophysical Techniques Aims and Assessment Nov/Dec 2008

FORMAT FOR LAB WRITEUPS. Include the following sections in your reports:

I. Background/Introduction∙ Purpose of the experiment. ∙ Important background and/or theory. ∙ Description of specialised equipment. ∙ Justification of experiment's importance.

II. Methods∙ Briefly describe procedure and any modifications to procedure (don't rewrite lab script, nor say 'see laboratory script').

III. Experimental Results∙ Present data in tables and graphs (don't forget to label all axes, number figures, and provide titles). ∙ Use sentences to draw attention to key points in tables or graphs.∙ Provide sample calculations. ∙ State key result in sentence form.

IV. Discussion∙ This is the most important part of the report, where you can show your understanding of the experiment. Discuss the significance or meaning of the results.∙ Analyse and interpret results and analyse experimental error.∙ Answer questions posed in lab.

V. Conclusion ∙ Very brief - did the experiment work and what did you learn?

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CH921: Biophysical Techniques Basic Lab Skills: Nov/Dec 2008

BASIC LABORATORY SKILLS

Introduction. Molecular biology, biochemistry and analytical chemistry all require a very high standard of basic laboratory skills if reagents and instrument time are not to be wasted. It is generally assumed that the limiting aspect of an analysis is the equipment used not the operator’s laboratory expertise. However, it is often the other way round. This session is designed to ensure you are not the limiting factor. Do not assume that because you have used a particular technique in an undergraduate course that you are sufficiently proficient. You will need to be checked off at each stage as indicated. A demonstrator must be convinced you have satisfactorily completed each task. You will have to have this laboratory session signed off before starting the next laboratory sessions.

Aims. The aims of this excercise are to ensure competency in the use of a 4 figure balance, calibration and use of micropipette, glass volumetric pipette, graduated glass pipette, measuring cylinder, and volumetric flask. Glassware is more expensive than you probably realize so take care.

You will need to bring to the laboratory: A4 laboratory notebook (get from chemistry stores) pocket sized notebook laboratory coat permanent OHP marker pen (for glassware, from chemistry stores) normal pen safety glasses (from chemistry stores)

You also must prepare safety data forms (before coming to the laboratory) for all compounds used. YOU ARE NOW RESPONSIBLE FOR THIS. You must read the chemistry department safety booklet before coming to the laboratory.

Part A. Use of 4-figure balance. Ask a demonstrator to show you how to use, calibrate, tare etc. a balance.

Part B. Calibration of pipettes and volumetric glassware.

Micropipette. Some of the micropipettes have been recently calibrated and are within 1-2%, others have not, so this is a dual function exercise. Select a P1000, a P200 or P100, a P20 or a P2. Examine each pipette and familiarize yourself with its mode of action. Each student must use 3 pipettes. Read the pipette manual before starting. To set a volume, wind to a slightly higher value than you want then wind down to the required value. If you overshoot, rewind to a higher value etc. Make sure the tip is firmly attached. Depress the plunger to the first stop, immerse tip of tip below the liquid surface (but not right to the bottom) and slowly suck up liquid (if you get air bubbles in it eject and start again). Remove the pipette from the liquid. You have now wetted the tip. It is best practice to eject this aliquot either to waste or back into the sample (consider waste versus contamination issues) by pressing the plunger to the second stop and repeat the sucking process. Look how much liquid is in your tip (your eye is surprisingly accurate). Eject the liquid into a container, if possible with the tip of the tip against the side of the container and the pipette held vertical. Repeat this a few times to practise. NEVER USE A MICROPIPETTE FOR ANYTHING

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EXCEPT water, alcohols (but check calibration), acetonitrile and innocuous aqueous buffer solutions. Check.

CH921: Biophysical Techniques Basic Lab Skills: Nov/Dec 2008

Calibrate the pipette (and your pipetting) by taking a beaker, placing it on the balance, taring the balance, and pipetting 1000 µL or 100 µL etc., as appropriate, of water into the beaker. Record the mass of water in your lab book. Repeat this process until you have 10 successive aliquots of exactly (to within the pipette's specifications) the same mass each time. Then re-tare and pipette 10 aliquots into it in quick succession. Weigh the total and determine whether the pipette is accurate or not. CHECK YOUR PIPETTING METHOD WITH A DEMONSTRATOR AND GET SIGNED OFF. Check.

Prepare an Excel spreadsheet showing the mean, standard deviation and relative standard deviation of your 10 individual aliquots for each pipette. Also note the mass of the 10× aliquots.

Volumetric pipette. Repeat the above with one glass pipettes using a pipette filler. THE VALVES ON THE FILLERS ARE DELICATE — TAKE CARE. Check with a demonstrator that you know what you are doing. Note. The pipettes are calibrated to have the bottom of the meniscus on the line of the pipette. Check.

Graduated pipette. Ensure you understand the markings on a graduated glass pipette.

Measuring cylinder. Repeat the above for a measuring cylinder.

Volumetric flask. Repeat the above for a volumetric flask.

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CH921: Biophysical Technique Introduction to Protein Databases Workshop Nov/Dec 2008

INTRODUCTION TO PROTEIN DATA BASES

SWISS PROT: Swiss Prot is a protein sequence database that was established in 1986 and is maintained by the Department of Medical Biochemistry at the University of Geneva and the European Bioinformatics Institute, EMBL data library.

Swiss Prot gives information on the function(s) of the protein; protein-translational modification(s); domains and sites; secondary and quarternary structures; similarities to other proteins; sequence conflicts and variants; and disease(s) associated with deficiency(s) in the protein. There is a high level of integration with other biomolecular databases.

To enter Swiss Prot use the following website link to the Sequence Retrieval System homepage:

http://us.expasy.org/srs5/

Start a new SRS session Select SWISS PROT (and deselect TREMBL if it is ticked) and press continue In order to search the database it is necessary to define some fields. It is useful to supply

as much information as is known about the protein. For example searching under Lysozyme alone reveals 121 hits.

Useful fields to complete include: Description; Organism from which the protein is from; Keywords or all text. From the search results choose the most suitable hit, by selecting this general

information can be found. The primary accession number is a number that will never change, so this can be

transferred to different databases.

ProtParam. To obtain data on chemical and physical properties for a given protein found using SWISS PROT, a tool called the ProtParam tool can be used. This can be accessed by using the following website:

http://us.expasy.org/tools/protparam.html

By entering the primary accession number the corresponding proteins sequence will appear. From this select the region that is the main chain. This will give information about the number of amino acids; molecular weight; amino acid composition; chemical formula and extinction coefficient with and without disulfide bonds amongst other information.

Protein DataBank. For information on the structure of a protein use the Protein Data Bank, web link:

http://www.rcsb.org/pdb/

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Once again enter the primary accession number and select find a structure. Many structures may appear, select the one with the highest resolution, and click on explore. Record the PDB code, this is the four digit code at the start of each entry.

CH921: Biophysical Technique Introduction to Protein Databases Workshop Nov/Dec 2008

A sequence details page appears, from this display the file by selecting Download/Display file. Display the complete file in text format. With less well known proteins it is important to search for any missing residues. To do this use find under edit on the main toolbar and search for MISSING.

Using this database it is possible to obtain sequence details and view the proteins structure.

PDBSum. Another useful database for viewing a proteins structure is:

http://www.biochem.ucl.ac.uk/bsm/pdbsum/

Enter the PDB code to view the structure.

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CH921: Biophysical Techniques Dichroweb CD Data Fitting Workshop: Nov/Dec 2008

DICHROWEB

Dichroweb is an online circular dichroism analysis facility.

www.cryst.bbk.ac.uk/cdweb

Select START ANALYSIS Login Enter file information. The file format is Jasco 1.50 if using data as a .txt file directly from the CD machine, if

converted to delta epsilon use free format (the answer you get ought to be the same!). The input units are machine units. Analysis programs - SELCON and CONTIN may be the best choice Reference set — use the best to fit your data. Output format – default, and output units - machine units. Submit the form. Protein concentration can be calculated using found in the protein databases and A280

Mean residue weight can also be calculated using the molecular weight and number of residues.

Work to do for each protein. From the protein databases find out the extinction coefficent; molecular weight; number

of residues and the protein sequence. Use dichroweb to generate plots using ONE fitting program e.g. SELCON. In excel plot your original experimental data in machine units and as a delta epsilon plot

(in terms of amino acids). Also plot dichroweb experimental and fitted data. Compare your experimental data with the dichroweb data. Record the percentage

secondary structure of your protein.

* Note your username and password here (there will be provided to you):

Username:Password:

Practice data set (monoclonal antibody fragment):dE=dA/(c*l) conc/mg/mL= 0.744pathlength/cm = 0.009185 MW= 27195 Daconcentration/uMaa= 0.0069763 no.aa= 255

Wavelength CD/mdegCD/mdeg zeroed HT delta A delta E

260 0.0983666 0.0135253 223.134 4.1E-07 0.0064002

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259 0.13072 0.0458787 223.398 1.39E-06 0.0217099258 0.070691 -0.01415 224.001 -4.3E-07 -0.006696257 0.0415099 -0.043331 224.634 -1.3E-06 -0.020504256 0.0667098 -0.018131 225.243 -5.5E-07 -0.00858255 0.103352 0.0185107 225.838 5.61E-07 0.0087593254 0.103459 0.0186177 226.454 5.65E-07 0.0088099253 0.052049 -0.032792 227.06 -9.9E-07 -0.015517252 0.0608287 -0.024013 227.666 -7.3E-07 -0.011363251 0.0720854 -0.012756 228.295 -3.9E-07 -0.006036250 0.104696 0.0198547 228.938 6.02E-07 0.0093953249 0.091961 0.0071197 229.573 2.16E-07 0.0033691248 0.0626229 -0.022218 230.22 -6.7E-07 -0.010514247 0.0540286 -0.030813 230.894 -9.3E-07 -0.014581246 -0.011942 -0.096783 231.606 -2.9E-06 -0.045798245 -0.080257 -0.165098 232.361 -5E-06 -0.078125244 -0.026234 -0.111075 233.15 -3.4E-06 -0.052561243 -0.008666 -0.093507 234.002 -2.8E-06 -0.044248242 -0.109426 -0.194267 234.962 -5.9E-06 -0.091928241 -0.133588 -0.218429 236.02 -6.6E-06 -0.103361240 -0.129282 -0.214123 237.199 -6.5E-06 -0.101323239 -0.108148 -0.192989 238.525 -5.9E-06 -0.091323238 -0.208626 -0.293467 239.958 -8.9E-06 -0.138869237 -0.243206 -0.328047 241.459 -9.9E-06 -0.155232236 -0.326162 -0.411003 243.029 -1.2E-05 -0.194487235 -0.344105 -0.428946 244.669 -1.3E-05 -0.202978234 -0.356551 -0.441392 246.382 -1.3E-05 -0.208868233 -0.587904 -0.672745 248.138 -2E-05 -0.318344232 -0.72738 -0.812221 249.92 -2.5E-05 -0.384344231 -0.683335 -0.768176 251.698 -2.3E-05 -0.363502230 -0.97801 -1.062851 253.46 -3.2E-05 -0.502943229 -1.19698 -1.281821 255.201 -3.9E-05 -0.60656228 -1.38729 -1.472131 256.915 -4.5E-05 -0.696615227 -1.6364 -1.721241 258.533 -5.2E-05 -0.814494226 -1.68129 -1.766131 260.075 -5.4E-05 -0.835736225 -1.87631 -1.961151 261.578 -5.9E-05 -0.92802224 -2.092 -2.176841 263.048 -6.6E-05 -1.030085223 -2.36917 -2.454011 264.513 -7.4E-05 -1.161242222 -2.57345 -2.658291 265.992 -8.1E-05 -1.257907221 -2.73054 -2.815381 267.472 -8.5E-05 -1.332243220 -2.83816 -2.923001 268.95 -8.9E-05 -1.383169219 -3.04081 -3.125651 270.407 -9.5E-05 -1.479063218 -3.12965 -3.214491 271.888 -9.7E-05 -1.521102217 -3.14932 -3.234161 273.436 -9.8E-05 -1.53041216 -3.13956 -3.224401 275.079 -9.8E-05 -1.525792215 -3.21076 -3.295601 276.821 -1E-04 -1.559484214 -3.13064 -3.215481 278.656 -9.7E-05 -1.521571213 -3.02681 -3.111651 280.683 -9.4E-05 -1.472438212 -2.94559 -3.030431 283 -9.2E-05 -1.434005211 -2.75135 -2.836191 285.571 -8.6E-05 -1.34209210 -2.66393 -2.748771 288.438 -8.3E-05 -1.300723

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209 -2.4253 -2.510141 291.636 -7.6E-05 -1.187803208 -2.14786 -2.232701 295.25 -6.8E-05 -1.056518207 -1.97766 -2.062501 299.33 -6.3E-05 -0.975979206 -1.72507 -1.809911 303.87 -5.5E-05 -0.856453205 -1.3651 -1.449941 308.96 -4.4E-05 -0.686114204 -1.00368 -1.088521 314.671 -3.3E-05 -0.51509203 -0.59642 -0.681261 320.966 -2.1E-05 -0.322374202 -0.126838 -0.211679 327.857 -6.4E-06 -0.100167201 0.295065 0.2102237 335.361 6.37E-06 0.0994782200 0.537299 0.4524577 343.46 1.37E-05 0.2141037199 0.840225 0.7553837 351.963 2.29E-05 0.3574487198 1.11442 1.0295787 360.738 3.12E-05 0.4871982197 1.57238 1.4875387 369.644 4.51E-05 0.7039056196 2.41985 2.3350087 378.579 7.08E-05 1.1049297195 2.86705 2.7822087 387.588 8.44E-05 1.3165454194 2.56803 2.4831887 396.939 7.53E-05 1.1750487193 2.78953 2.7046887 406.767 8.2E-05 1.2798628192 3.23624 3.1513987 416.093 9.56E-05 1.4912467191 2.558 2.4731587 426.171 7.5E-05 1.1703025190 2.68676 2.6019187 436.241 7.89E-05 1.2312319

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CH921: Biophysical Techniques Experiment I: Nov/Dec 2008

EXPERIMENT I:REAGENT AND BUFFER PREPARATION

Reagent preparation. You will be allocated solutions from the following list to make up. Determine which experiment the reagent is used in (you will have to read ahead in your lab manuals) and calculate how much of the reagent each person will require. To determine how much total solution to make, multiply by a factor of class number times 1.25.

1.1 200 M [Ru(1,10-phenanthroline)3]2+ in water

1.2 100 mM NaCl in water

1.3 50 mM phosphate buffer, pH=7. (4 people) (NB don’t waste solutions you will need to make up much more of one component than the other)

1.4 2 mg/mL protein standard solution

1.5 Biuret reagent: Place CuSO4.5H2O (1.5 g) and sodium potassium tartrate.4H20 (6.0 g) into a dry 1 L volumetric flask add about 500 cm3 of water. With constant swirling, add NaOH solution (300 mL, 10% w/v). Make to volume (1L) and mix. The reagent prepared in this manner is a deep blue. It may be stored indefinitely if KI (1 g) is also added and the reagent is kept in a plastic container.

1.6 Dissolve Coomassie Brilliant Blue G-250 (100 mg) in 50 mL 95% ethanol. To this solution phosphoric acid (100 cm3 85% w/v) is added and the solution diluted to 1 L. (Alternatively, a pre-prepared dye reagent concentrate can be purchased from Bio-Rad and diluted by adding 4 volumes of distilled water to 1 volume of concentrate.)

Buffers. The following tables describe how two important buffers, acetate and phosphate buffer, are prepared at a range of pH values by mixing different amounts of two stock solutions. (From Methods in Enzymology, Vol. 1, p.138)

Acetate buffer. Stock solutions: A: 0.2 M solution of acetic acid (11.55 mL in 1000 mL H2O) B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or

27.2 g of C2H3O2Na.3H2O in 1000 mL.

(mL A + mL B, diluted to a total of 100 mL)

A (mL) B (mL) pH A (mL) B (mL) pH46.3 3.7 3.6 20.0 30.0 4.844.0 6.0 3.8 14.8 35.2 5.041.0 9.0 4.0 10.5 39.5 5.236.8 13.2 4.2 8.8 41.2 5.430.5 19.5 4.4 4.8 45.2 5.625.5 24.5 4.6

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CH921: Biophysical Techniques Experiment I: Nov/Dec 2008

Phosphate buffer. Stock solutions: A: 0.2 M solution of monobasic sodium phosphate (27.8 g in 1000 mL H2O).

B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4

.7H2O or 71.7 g of Na2HPO4.12H2O in 1000

mL H2O).

(mL A + mL B, diluted to a total of 200 mL)A (mL) B (mL) pH A (mL) B (mL) pH93.5 6.5 5.7 45.0 55.0 6.992.0 8.0 5.8 39.0 61.0 7.090.0 10.0 5.9 33.0 67.0 7.187.7 12.3 6.0 28.0 72.0 7.285.0 15.0 6.1 23.0 77.0 7.381.5 18.5 6.2 19.0 81.0 7.477.5 22.5 6.3 16.0 84.0 7.573.5 26.5 6.4 13.0 87.0 7.668.5 31.5 6.5 10.5 90.5 7.762.5 37.5 6.6 8.5 91.5 7.856.5 43.5 6.7 7.0 93.0 7.951.0 49.0 6.8 5.3 94.7 8.0

Questions (answers to be submitted).

1. How does a buffer work? What determines its pH range?2. Why might you need to use acetate rather than phosphate?3. What is the concentration of sodium in the standard pH = 7.2, pH = 7.0 and pH = 6.8 phosphate buffer?4. Write brief notes on buffers. Consider a pH=7 buffer. If 1 mL of a 100 mM (in phosphate) stock solution is used, how much buffering can this solution do? Is it likely to be enough for a solution of 1 mL of 2 mg/mL protein?

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CH921: Biophysical Techniques Experiment II: Nov/Dec 2008

EXPERIMENT II:PREPARATION OF DNA AND PROTEIN SAMPLES

Aim. The aim of this experiment is to prepare the DNA and proteins samples which you will use in Experiments 3-5. Before weighing out your DNA or protein you must read ahead in the lab script and calculate the amounts of these substances you will need (this is discussed in more detail in Parts A and B).

Part A: DNA Sample preparation. Determine how much DNA you require to make up 5 mL of a 1400 M in base DNA solution. (Take a base to have average molecular weight of ~330 Da.) Get this checked with a demonstrator. Once approved, weigh out this amount of calf thymus DNA and add 4 mL of water. Make sure the DNA is beginning to hydrate (it will start to look translucent) then leave it in the refrigerator over night. Calf thymus DNA is sufficiently stable that we can get away without buffering it for this experiment.

Part B: Protein sample preparation. You will be making up your protein sample (US) to be ~ 1 mg/mL. But note that this will not be an accurate guide to concentration as it may not be pure but contaminated with salts or nucleic acids. Determine how much stock solution you will need to run a circular dichroism spectrum (200 L of 0.1 mg/mL and 60 L of 1 mg/mL) and to do the required protein concentration determination experiments. Construct a table with the required volumes of 1 mg/mL stock needed for each assay, and get this checked with a demonstrator. Once approved, make up this solution in water. If the protein is reluctant to dissolve you may need to add a drop of dilute (~ 1 M or less) acid (usually one chooses HCl).

Protein List: Lysozyme Ribonuclease A -Lactalbumin Myoglobin -Chymotrypsin Cytochrome c

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CH921: Biophysical Techniques Experiment III: Nov/Dec 2008

EXPERIMENT III:COMPARISON OF THREE METHODS FOR THE ESTIMATION OF PROTEIN

CONCENTRATION

Aims. For a variety of purposes, including all structural studies of proteins and in order to determine the specific activity of an enzyme at different stages of purification, one must have a sensitive method for estimating protein concentration. In this experiment you will compare three different methods and evaluate their relative merits.

Solutions. At this point, you will have two protein stock solutions which you will use throughout Experiment III. The first is the protein stock solution you made in Experiment II, referred to here as unknown protein stock (or US) and having a concentration of approximately 1 mg/mL. The second protein stock solution is that of a protein standard stock (denoted SS), whose concentration is accurately known and given on the bottle (~2 mg / mL). With each method you will need to dilute the stock to the appropriate concentration range for the assay. Note in your laboratory book and your report what (weighing, measuring volumes etc. in a table) you have done to make the solutions you have used. In each case perform repeat measurements on each unknown sample.

Calibration curves. For Assays B, C, and D, you will need to plot calibration curves using results from a protein of known concentration (made from SS). Plot absorbance verses µg of protein in the assay mixture. You may plot the data electronically but a plot on graph paper will be required for your assessment. Use your curve to determine the µg of U in your assay mixtures by drawing a horizontal line from the absorbance reading of the unknown to the calibration curve, then dropping a vertical line to read the µg of protein in the mixture. Hence determine the concentrations in the stock solutions.

Assessment. In addition to standard requirement, please include the following in your report.

Plot standard curves using the data for the known concentrations. Determine the U concentrations using each method. Discuss your results in terms of the relative sensitivity and accuracy of the four methods. Comment on the errors in the measurements.

Outline the chemistry of each method.

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CH921: Biophysical Techniques Experiment III-A: Nov/Dec 2008

ASSAY A: ABSORBANCE AT 280 NM.

In this assay you will measure the A280 of the approximately 1 mg/mL US protein sample in quartz cuvettes. You will then use this information, along with the extinction coefficient determined in the data base exercise, to calculate concentration.

Protocol. Put ~ 2 mL 18.2 M water in either a clean dry or clean water rinsed quartz absorbance cuvette. Set the instrument parameters (in file menu on V-550 or V-570) to collect data from 400 - 200 nm in 0.5 nm steps with a response time of ~ 400 nm/min (fast). Run a baseline/background spectrum with the cuvette in the sample position.

Take a clean dry 1 cm quartz absorbance cuvette (you may need to wash an old sample out by emptying the cuvette, filling it with water, emptying it - repeat at least 2 times). Then fill with acetone, empty, repeat 2 times. Dry cuvette either with nitrogen line (or air if nitrogen is not available) or a hair dryer. Pipette directly into the cuvette: 1 mL of your US; add 1 mL 18.2 M water.

On the V-550/570 select autozero from the file menu. Measure a spectrum using the same parameters as the baseline. Save your data directly onto a floppy disk as jws format and txt format if you are using the V-550/570.

Determine for your protein from its amino acid sequence (see computer session for how to get this information or calculate it from the sequence as indicated in lectures)*. Use the Beer Lambert Law to determine the concentration of your US. Compare this value with that obtained by assuming that a 1 mg/mL solution has an absorbance of 1.0. Comment on any differences. What is the assumption underlying this method? Compare both values for concentration with that obtained from the equation: 1.55 A280 - 0.76 A260 = mg protein/mL. Comment. What is the rationale behind this equation? (*cystine, max~120 mol1dm3cm1; Tyr, max ~ 1280 mol1dm3cm1; Trp, max~ 5690).

Exercise. If lysozyme has a molecular weight of 14314, nW = 6, nY = 3, nC = 8, determine its . Compare this with the experimental value of 280 = 37932 mol1dm3cm1. The values for chymotrysinogen are: 25670, 8, 4, 10, 51340. Comment.

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CH921: Biophysical Techniques Experiment III-B: Nov/Dec 2008

ASSAY B: BIURET METHOD.

(Reference: Gornall, Bardawils and David, J Biol Chem 1949, 177, 751)

This method is simple and reasonably specific as it depends on the reaction of copper (II) with N atoms in the peptide bonds of proteins. Compounds containing peptide bonds give a characteristic purple colour when treated in alkaline solution with copper sulfate. This is termed the 'biuret' reaction because it is also given by the substance biuret NH2—CO—NH CO—NH2, a simple model compound.

C

O

C H

N N C

H R H O

C u ( I I )

C

O

C H NNC

HRHO

For a wide variety of proteins, 1.0 mg of protein in 2 mL of solution results in an OD at 540 nm of 0.100. This assay is sensitive to 0.5 – 2.5 mg protein in the assay mixture

Many haemoproteins give spurious results due to their intrinsic absorption at 540 nm, but modifications which overcome this difficulty are known (either removal of the haem before protein estimation or destruction of the haem by hydrogen peroxide treatment). The protein content of cell fractions such as nucleii and microsomes can be estimated by this method after solubilisation by detergents such as deoxycholate or sodium dodecyl sulphate.

Protocol. You will begin by preparing SS standards for a calibration curve containing: 0.0 mg protein; 0.5 mg protein (e.g. 250 µL SS solution); 1.0 mg protein; 1.5 mg protein; and 2 mg protein from the 2 mg / mL SS protein solution provided. Also prepare duplicates of 2 different concentrations of US (US should always be measured in duplicate). To prepare the protein solutions, mix the protein solution (x µL, where x < 1500 µL) with water [(1500 - x) µL] to make a total volume of 1500 µL. Summarize your calculations in a table and have it checked by a demonstrator. Once approved, prepare your samples.

Add 1500 µL biuret reagent and mix. The purple colour is developed by incubating for 15 minutes at 37C. Cool the tubes rapidly to room temperature. Measure a spectrum of this solution and also of a reference solution containing 1500 µL biuret reagent and 1500 µL water, which has also been incubated at 37C. Samples to be measured should be at room temperature and should not be unduly warm or ice cold because the colour intensity of the copper complex has a high temperature coefficient. Read the absorbances at 540 nm. The colour of the solutions is stable for hours. Plot a calibration curve using protein standard and use the curve to determine the concentration of US. * Important note: the Beer-Lambert Law does not hold for these solutions at optical densities above 0.25.

Relatively few substances interfere with the biuret estimation; those which do, include bile pigments, sucrose, tris, glycerol, imidazole and ammonium ions. Sucrose, tris and glycerol can usually be corrected for by their inclusion in the blank and protein standard.

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CH921: Biophysical Techniques Experiment III-C: Nov/Dec 2008

ASSAY C: COOMASSIE BLUE DYE BINDING ASSAY.

(References: MM Bradford, Analytical Biochemistry 1976, 72, 248; SM Read, DH Northcliffe, Anal Biochem 1981, 96, 53.)

This protein determination method involves the binding of Coomassie Brilliant Blue G250 to protein. The protonated form of Coomassie Blue is a pale orange-red colour whereas the unprotonated form is blue.

When proteins bind Coomassie Blue in acid solution their positive charges suppress the protonation and a blue colour results. It has been found that hydrophobic interactions between the dye and the protein are very important in the binding process. The binding of the dye to a protein causes a shift in the absorption maximum of the dye from 465 to 595 nm and it is the increase in absorbance at 595 nm which is monitored. The assay is very reproducible and rapid with the dye binding process virtually complete in ~ 2 minutes with good colour stability.

The only compounds found to give excess interfering colour in the assay are relatively large amounts of detergents such as sodium dodecyl sulphate, Triton X-100 and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls. The assay is non-linear and requires a standard curve. The standard assay described below is useful for protein solutions containing 10 to 100 µg of protein in a volume up to 100 µL. (The micro-protein assay described in Bradford's article can be used for protein solutions containing 1 to 10 µg proteins in a volume up to 100 µL, but requires the use of a microcuvette.)

Protocol. You will again begin by preparing SS standards for a calibration curve containing: 0 µg, 20 µg, 40 µg, 60 µg, 100 µg of protein from the 2 mg / mL SS protein solution provided. Also prepare duplicates of 2 different concentrations of US. Because this assay is very sensitive (sensitivity 20 – 140 µg protein), you will need to prepare more dilute (1/5 th concentration) standard solutions from SS and US.

Place required volumes, x µL, of the 1/5 th concentration standard solutions in clean, dry test tubes. Add (500 – x) µL water. Also place 500 µL water or sample buffer in "blank" test tubes. Add 5.0 mL diluted dye reagent (Bio-Rad) to each sample. Vortex (avoid excess foaming) or mix several times by gentle inversion of test tube. After a period of from 5 – 60 minutes, determinine A595. Plot A595 versus the amount of protein in each assay tube. Read unknowns from the standard curve.

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CH921: Biophysical Techniques Experiment III-D: Nov/Dec 2008

ASSAY D: BCA METHOD

A standard solution of a chosen standard protein (usually 2.00 mg/mL bovine serum albumin) should be created. In this case the table below summarises the protein solutions that should be made for the calibration curve. Two U sample concentrations should be chosen to give values in the middle of the calibration graph. Repeat the measurement of each U concentration.

Protein concentration(g/mL)

L 2 mg/mL BSA solution

L buffer or water

1000 50 50500 25 75200 10 9050 2.5 97.50 0 100

Protocol. Make a stock reaction mixture solution by mixing 20 mL BCA reagent, Pierce No. 23223 and 285 L 4% CuSO4. For each analysis: make 100 L protein solution of the desired concentration in buffer or water. Mix well. Add 2 mL of the reaction mixture from step 1. Mix well. Incubate at 37C for 30 minutes.

Allow the tubes to cool down to room temperature and then measure the absorbance at 562 nm having zeroed the spectrometer on a water sample. Plot the readings for each standard as a function of protein concentration. Use the resulting curve to determine the concentration of the unknown protein.

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CH921: Biophysical Techniques Experiment IV: Nov/Dec 2008

EXPERIMENT IV:PROTEIN SECONDARY STRUCTURE DETERMINATION BY CIRCULAR DICHROISM

Protocol. The practical part of this experiment is straight forward and requires a 0.1 mg/mL solution of your US protein to be put into a 1 mm quarz cuvette and a CD spectrum collected from 260 – 190 nm. A buffer baseline (which will already have been run by the demonstrator) needs to be subtracted to give you the sample’s CD spectrum. Repeat the experiment with 1 mg/mL US protein solution and a 0.1 mm demountable cell.

Parameters should be: 100 nm/s; response time = 1 s; data interval = 1 nm; bandwidth = 2 nm; accumulations = 4. Wash your cuvette with water (at least 3 times) and acetone (3 times). Dry it with a hair dryer or nitrogen line.

You should save your data files (sample and baseline) as a txt files for analysis. Use Excel to subtract the baselines and plot the CD spectra of the proteins (both in mdeg and ). You will need in addition to your spectrum a reasonably accurate molar concentration of your protein solution. Ideally this will come from Experiment III or Experiment III as revised during the analysis session. Determine the -helical content of your protein as given below and compare the answer with that from the crystallographic data base.

IAMBEC: assume that 100% -helical protein has 208 nm ~ -12 mol-1 dm3 cm-1. MOAC: Use Curtis Johnson’s CD structure fitting program CDsstr.

CD structure fitting data analysis using CDsstr for far UV spectra. For each sample that has been measured for which CD structure fitting is required, take the text file for the baseline substracted and zeroed spectrum and edit it in Excel or another piece of software to produce the data in the following form: One title line containing anything, followed by 71 lines (assuming fitting is being undertaken from 260 nm to 190 nm) of numbers corresponding to the CD spectrum in units of moles of (amino acids)-1 dm3 cm-1 with only two decimal places. If you have more than one data set, the second set starts on the line directly below the first.

nsure that the CDsstr program and the required associated files are located in a directory on the C drive of the computer you are using. The files include: procd190.tst; readme.cd; secstr.dta; bascd.dta; Cdsstr.exe. procd190.tst is a data file that can be used to test the program; it has three data sets in it.

To run the program, proceed as follows. Delete, rename, or move any file with a .out filename extension remaining in the CDsstr folder. Delete any previously used file named proCD.dta unless you wish to use it in the current run. If it is not already available, prepare an input file called procd.dta containing the CD data of the protein(s) to be analyzed. Save the file as c:\cdsstr\proCD.dta. Begin the analysis by opening a DOS window within windows. Type ‘c:’. Then type ‘cd\cdsstr’ at the command prompt. Type ‘cdsstr’ to run the program. Enter values for the program variables as prompted. NbasCD = 22; Nwave = 71; Npro = number of data sets in procd.dta; ncomb = 100; icombf = 100000. When the command prompt reappears, view, print and record in the laboratory book the results of the analysis by inspecting the output files anal.out and reconCD.out.

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CH921: Biophysical Techniques Experiment V: Nov/Dec 2008

EXPERIMENT V:EQUILIBRIUM BINDING CONSTANT DETERMINATION BY FLUORESCENCE

SPECTROSCOPY: DNA AND [RU(1,10-PHENANTROLINE)3]2+

This experiment is referred to as a titration series but you will save instrument time by preparing all the solutions before hand. (If the DNA is very expensive then you can save sample by adding DNA successively to the cuvette and simultaneously adding the same volume of ligand at twice the cuvette concentration. Alternatively you can dilute a concentrated DNA cuvette by adding ligand at the appropriate concentration.) Each solution will have 10 M of Ru and a varying amount of DNA: 0, 10 M, 20 M, 40 M, 80 M and 300 M.

Protocol. Determine the concentration of your DNA stock solution by measuring the A260 for a ~100 M DNA in base solution by measuring the absorbance spectrum from 320 - 200 nm. 258(calf thymus DNA) = 6600 mol-1 dm3 cm-1. Repeat the measurement (at least twice) until a consistent value is obtained.

Construct a table of volumes of stock solutions (including water) required to enable you to run the experiment at the above DNA and Ru concentrations with 10 mM NaCl and 1 mM (pH=7) phosphate buffer. Total volumes should be 3 mL. Check your table with a demonstrator.

Make up one Ru solution, 5 containing Ru and DNA and one containing only buffer and water. Collect fluorescence spectra for the seven samples. Save the data to disk as txt files. Determine the concentrations of bound and free Ru in each case and use the data to perform a Scatchard plot as outlined in lectures.

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CH921: Biophysical Techniques DNA Melting Curve Exercise: Nov/Dec 2008

DNA MELTING CURVE. Determination of thermodynamics of the helix to coil transition

INTRODUCTION. Under normal physiological conditions DNA exists as a double helix with two polymers held together by hydrogen bonds between the bases on each strand. Most biological processes involving DNA (including transcription, replication) require the DNA to separate into single stranded components. We can model this process to determine aspects of its thermodynamics by taking a solution of double stranded (ds) DNA helices and gradually heating them up until each DNA duplex has separated into two single stranded (ss) pieces of DNA that are often described as random coils. The so-called melting temperature of a DNA helix, Tm, is defined to be the temperature at which half the DNA base pairs have broken their hydrogen bonds. As the melting is cooperative (a single molecule unzips once it has started) this is usually equivalent to half the DNA molecules being ds and half ss.

Duplex DNA has the DNA bases held together in a fairly rigid arrangement with each base stacked between the bases above it and below it and hydrogen bonded to a base in the other strand. When the ds to ss transition takes place, the bases move much more freely and the base-base stacking is essentially removed. This means the π electrons of one base are no longer interacting strongly with those of the neighbouring base. The removal of π-π stacking causes the UV absorbance of the bases at about 260 nm to increase in magnitude. We can therefore monitor the transition by measuring the absorbance of a DNA sample as a function of temperature. You will have two columns of data. The first column is the temperature at which a measurement is taken, T, and the second column is the absorbance of a piece of DNA at that temperature, A. The following series of calculations will enable you to determine H, S, and G for the helix to coil transition.

1. Plot A versus T. (i.e. take A as the y axis and T as the x-axis). Estimate Tm by using a ruler to determine the point half way between the low T A curve and the high T curve. The value you determine is only approximate if the baseline of the curve is not flat.

2. Differentiate the A curve with respect to T. The maximum of this plot is another approximate value for Tm.

3. Using a ruler draw a straight line through the first 15 degrees of data points. This line approximates the temperature dependence of the unmelted DNA absorbance if you could hold it together. Work out an equation for this line and use this to create a column of data points that plot the straight line overlaid on top of your part 1 curve.

4. Determine the final absorbance on the melting curve plot, then evaluate

x

x y

as a function of temperature where x is the difference between the final absorbance of the unfolded DNA and the absorbance at a given temperature and y is the difference in absorbance between the temperature dependent absorbance and the dotted base line of your part 3 figure.

5. Create a temperature column in Kelvin, Tabs, and then create a 1/Tabs column. Plot versus the inverse of the absolute temperature (in Kelvin). The melting temperature is then the temperature at which half of the DNA is melted, i.e. the midpoint of the versus 1/T curve.

6. Plot the derivative of with respect to the inverse of the absolute temperature versus T. Determine the van't Hoff transition enthalpy of the transition

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HVH B'

(1 / Tmax ) (1/ T2 )

where Tmax is the absolute temperature of the maximum of the derivative curve you have just

plotted and T2 is the absolute temperature of the high temperature half height of the curve. B' = 4.38 cal K1 mol1; to convert from calories to Joules multiply by 4.1868 J/cal.

7. Determine the entropy change of the transition using

S HVH

Tm

8. Determine the Gibbs free energy of the transition and comment on the magnitudes of the three thermodynamic properties you have determined.

ReferenceL.A. Marky; K.J. Breslauer, Biopolymers, 1987, 26, 1601–1620

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