MOAC Doctoral Training Centre

______MOAC DOCTORAL TRAINING CENTRE NOVEMBER /DECEMBER 2012

CH921: TECHNIQUES FOR THE CHARACTERISATION OF BIOMOLECULES COURSE LEADER: DR. ANN DIXON Contributors: Prof. Alison Rodger; Prof. Peter O'Connor; Prof. Steven Brown; Dr. Claudia Blindauer; Dr. Vilmos Fulop; Dr. Jozef Lewandowski, Dr. Ivan Prokes

TABLE OF CONTENTS ______PAGE Deadlines and other important information 3 Timetable 4 Essay assignment 5 Laboratory and workshop manual: Workshop 1 6-7 Protein List 8 Workshop II 9 Aims and Assessment 10 Guidelines for preparation of lab report 11 Experiment I 12-13 Experiment II 14-18 Experiment III 19

2 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 Sarah Shute 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.  As:MIT students to submit all work to Christina Forbes.  MOAC students to submit all work to Sarah Shute.

 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. Marks will be deducted from late work (1% / hour late; 5%/day late) unless an extension is granted in writing from Dr. A. Dixon.

 Workshop problems/proof of completion: Due by 5 pm on the day of the workshop.  Essay: Due Tuesday 20 November at 4 pm.  NMR assessment: Due Tuesday 4 December at 4 pm.  Laboratory reports: Due Tuesday 11 December by 4 pm.

An oral examination (with 2 hours written/reading work before hand, to be submitted at the oral) will take place on Monday 3 December. You may take up to one A4 sheet of handwritten notes only into the written part of the examinations.

3 BREAKDOWN OF MARKS ______ Assessed work: 45%  Exam: 45%  Attendance: 5%  Laboratory conduct: 5%

CH921 TIMETABLE AUTUMN 2012 ______WEEK 6: NOV 5 AND 6 Mon. 9:00-10:00: Introduction to techniques for the characterization of biomolecules (Dixon, MOAC Lecture Room) 10:00-12:00: Databases workshop (Dixon, MOAC Lecture Room) 13:00-17:00: Obtain Essay title and begin work on Essay, perform calculations required for Experiment I

Tues. 9:00-11:00: UV lecture and workshop (Rodger, MOAC Lecture Room) 11:00-13:00: Fluorescence lecture, & workshop, (DNA melting curve) (Rodger, MOAC Lecture Room) 14:00-17:00: Laboratory: Experiments I & II (Dixon, Chemistry B309)

WEEK 7: NOV 12 AND 13 Mon. 9:00-11:00: CD lecture (Rodger, MOAC Lecture Room) 11:00-12:00: CD workshop (Rodger, MOAC Lecture Room) 14:00-16:00: Linear Dichroism lecture (Rodger, MOAC Lecture Room)

Tues. 9:00-10:00: Pre-lab (Dixon, MOAC Lecture Room) 10:00-17:00: Laboratory: Exp. III (Dixon, Chemistry B309)

WEEK 8: NOV 19 AND 20 Mon. 9:00-12:00: Introduction to Mass Spectrometry (O'Connor, MOAC Lecture Room)

Tues. 9:00-13:00: Introduction to NMR (Brown, MOAC Lecture Room) 14:00-17:00: NMR Group Work (Brown, MOAC Lecture Room) *Deadline 1: Essay due (4 pm)

WEEK 9: NOV 26 AND 27 Mon. 9:00-12:00: Bio-applications of high field NMR (Blindauer, MOAC Lecture Room) 13:00-15:00: X-Ray crystallography lecture (Fulop, BSR5, Bio. Sci) 15:00-18:00: Crystallography demo (Fulop, BSR5, Biol. Sci.; until 6 pm)

Tues. NMR Demonstrations 9:30-12:30 (MOAC Students): (Lewandowski, Milburn House) 10:00-12:00 (As:MIT Students): (Prokes, Milburn House)

WEEK 10: DEC 3 AND 4

4 Mon. 9:00-17:00: EXAM

Tues. 10:00-11:00: Pre-lab (Dixon, MOAC Lecture Room) 11:00-16:00: Laboratory: Exp. V (Dixon, Chemistry B309) *Deadline 2: NMR Assessment due (4 pm)

WEEK 10+1: DEC 11 Tues. *Deadline 3: Lab reports & Feedback forms due

ESSAY ______Select one of the following essay topics:

1. Organisation and composition of (various) cellular membranes 2. The structures of membrane proteins 3. Sugar-binding proteins: Structure and Function 4. The structure of viral particles / viruses

Include in your essay a discussion of the structure of the system you have chosen. Also include how different biophysical techniques have been used to acquire the structural data. Conclude with a critical analysis of the techniques used including their advantages and disadvantages.

Write using an American Chemical Society Journal template, found at:

http://pubs.acs.org/page/jacsat/submission/authors.html and cite references using an ACS approved style - for a nice summary see:

http://library.williams.edu/citing/styles/acs.php

Use at least 10 primary recent literature references. Web pages are not included as references in this count but must be acknowledged if you use diagrams taken from websites.

Write 1500-2000 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, format etc.

Both an electronic and hard copy version should be submitted to Sarah Shute on or before the deadline of 4 pm, Tues. Nov. 20, 2012.

5 WORKSHOP I: INTRODUCTION TO PROTEIN DATA BASES

UNIPROT ______The Universal Protein Resource (or UniProt) is a comprehensive resource for protein sequence and annotation data. UniProt consists primarily of three databases: the UniProt Knowledgebase (UniProtKB); the UniProt Reference Clusters (UniRef); and the UniProt Archive (UniParc). UniProt is a collaboration between the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR) with the objective of the curation and organisation of huge numbers of protein sequences.

UniProt 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 UniProt, use the following website link to the Sequence Retrieval System homepage: http://www.uniprot.org/

 Select “UniProt KB” (UniProt knowledge base) from the “Search in” drop down menu, but note that there are other options available that you can explore in your own time.  In the “Query” box, type the name of a protein or other keyword. As a practice run, type “Ubiquitin”, then click “Search”.  You should have about 85,000 hits – that’s a lot of information about Ubiquitin. But you’ll need to narrow it down as you will most often be looking for information about a particular protein.  Click the “advanced search” link on the right-hand side of the screen, and type in another search term to narrow down the number of hits. In this case, we will search for Ubiquitin from a particular organism. Select “Organism” from the “Field” drop down menu, but again note that there are other options available that you could use to narrow your search. Then type “Human”, select from the list, and click “search”.  Now you should have over 3,000 hits – that is still a lot, but in this case the one you want is the first hit (primary accession number P0CG47, UBB_HUMAN). Note: The primary accession number is a number that will not change and is used for only one protein, and this can be transferred to different databases.  Select UBB_HUMAN by clicking on the accession number (P0CG47), and this will bring up the file on this particular protein. Scroll down and you will see a large variety of information about this protein, including key references, links and the protein sequence.

6  Go to the protein sequence and you will see a drop down menu labelled “Tools”. You will now use these tools to analyse the protein sequence.

PROTPARAM ______To obtain data on chemical and physical properties for a given protein found using UniProt, a tool called ProtParam can be used. This can be accessed by clicking “ProtParam” in the “Tools” drop down menu next to the protein sequence, and then clicking “go”. Alternatively, you can go directly to the ProtParam analysis website at:

http://expasy.org/tools/protparam.html and entering either the primary accession number or the sequence of the corresponding protein.

Once you have clicked “ProtParam” in the “Tools” drop down menu next to your protein sequence, select the region that is the main chain (1-76 in the case of Ubiquitin). 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.

RCSB PROTEIN DATA BANK ______For information on the structure of a protein use the Protein Data Bank, web link:

http://www.pdb.org/pdb/home/home.do

 You can search this database using the primary accession number by clicking the “Advanced Search” link on the upper right hand side of the screen.  In the Advanced Search interface, select “UniProtKB Accession Number(s)” in the “Choose a query type” drop down menu, and enter your accession number in the “Accession IDs” box. Then select “Submit query”.  You should get approximately 10 hits. To start, you should look for structures (a) of just the protein (no bound ligands, etc), and (b) with the highest resolution. For this example search, you could select the structures with the PDB IDs 2XK5, 3NOB, or 2XEW.  This will connect you to a page with several thumbnails at the top where you can see structure and sequence details as well as many other types of information. Have a look around, then view the structure using the .jmol viewer. You can save an image of the structure by right clicking and selecting the appropriate option for the file type you prefer. You can also download the structure file by selecting “Download Files”, and you can 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.

YOUR ASSIGNMENT ______You will be assigned a protein from the list on the following page. Search for and find the protein you have been assigned in the UniProt database as you just did above. In a separate document, you’ll need to include the following:

7 A. UniProt entry for that protein, including the protein sequence B. ProtParam analysis output for the protein C. An image of the X-Ray or NMR structure for that protein

When you are done, you’ll need to show your report to a demonstrator in order to be approved. This is a pass / fail workshop, and you will need to perform the exercises until you pass. You will use the information you collect in this workshop to help you complete the lab tasks (you will be using the same protein you were assigned here to perform your lab experiments), so you’ll need to bring a copy of your report to the lab.

PROTEIN LIST ______

 Lysozyme  Ribonuclease A  -Lactalbumin  Myoglobin  -Chymotrypsin  Cytochrome c

8 WORKSHOP II: FITTING OF CIRCULAR DICHROISM DATA

DICHROWEB ______Dichroweb is an online circular dichroism analysis facility. To access Dichroweb for analysis of CD data, use the following website link and then follow the instructions below:

www.cryst.bbk.ac.uk/cdweb

 Select START ANALYSIS  Login (you will be given a password for this program at the start of the module)  Enter file information.  The file format is Jasco 1.50 if using data as a .txt file directly from the CD machine (this will be in units of mdeg). If the data has been converted to units of delta epsilon, use the free format (the answer you get ought to be the same whichever units you use).  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 workshop) in conjunction with the A280 and Beers Law.  Mean residue weight can also be calculated using the molecular weight and number of residues.

YOUR ASSIGNMENT FOR EACH PROTEIN ______ From the protein databases, you should determine the extinction coefficient, molecular weight, number of residues, and the protein sequence.  Use dichroweb to generate plots using ONE fitting program e.g. SELCON.  In MS 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: ______

9 Password: ______

* Practice data set (monoclonal antibody fragment) available on CH921 website:

LABORATORY PRACTICALS

INTRODUCTION ______The aims of this lab course are to familiarize you with biological samples and teach you competence in a set of standard spectroscopic and analytical techniques. These include:  COSHH risk assessment, reagent preparation, and protein sample preparation (Experiment I).  Protein concentration determination (Experiment II).  Protein secondary structure determination by circular dichroism (Experiment III). 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. No extra make up session will be available.

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:  Improvement of laboratory skills with respect to sample handling  Tidiness and cleanliness  Accuracy of results  Care of equipment  Organization 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.

10  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.

FORMAT FOR LAB WRITEUPS ______Write up Experiments 1-3 as a single report. Reports should be NO LONGER than 15 pages with 1.5 spacing between lines and no smaller than 11 point Arial font. Points will be deducted for longer reports/smaller font. You will be marked on scientific content as well as use of correct spelling, grammar, formatting and clarity. Remember - someone has to read this!

I. Background/Introduction: Shows fundamental understanding of why we did the experiment, how the experiment works, and what the advantages/disadvantages are. ∙ 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).

III. Experimental Results ∙ Present data in tables and graphs (don't forget to label all axes, number figures, and provide titles). ∙ Refer to all tables and graphs in the text - i.e. use complete sentences to draw attention to key points in tables or graphs. ∙ Provide a sample calculation for each type of calculation. This is the only way to get partial credit for incorrect calculations. Also, for each calculation highlight the final answer in bold so I don't have to hunt around (and possibly not find) your answer. ∙ State key results in sentence form, and summarize results in tables. ∙ All figures must contain figure captions.

IV. Discussion ∙ This is the most important part of the report, where you can show your understanding of the results of the experiments. 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?

11 EXPERIMENT I: REAGENT AND PROTEIN SAMPLE PREPARATION

REAGENT PREPARATION ______You will be preparing a buffer solution and using it to prepare your US protein stock solution and all subsequent dilutions of both US and SS.

BUFFER PREPARATION ______The following table (Table 1) describes how an important buffer, phosphate buffer, is prepared at a range of pH values by mixing different volumes of two stock solutions. (From Methods in Enzymology, Vol. 1, p.138) Use Table 1 to prepare 100 mL of 50 mM phosphate buffer, pH=7.

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) Table 1: A (mL) B (mL) pH A (mL) B (mL) pH 93.5 6.5 5.7 45.0 55.0 6.9 92.0 8.0 5.8 39.0 61.0 7.0 90.0 10.0 5.9 33.0 67.0 7.1 87.7 12.3 6.0 28.0 72.0 7.2 85.0 15.0 6.1 23.0 77.0 7.3 81.5 18.5 6.2 19.0 81.0 7.4 77.5 22.5 6.3 16.0 84.0 7.5 73.5 26.5 6.4 13.0 87.0 7.6 68.5 31.5 6.5 10.5 90.5 7.7 62.5 37.5 6.6 8.5 91.5 7.8 56.5 43.5 6.7 7.0 93.0 7.9 51.0 49.0 6.8 5.3 94.7 8.0

PROTEIN SAMPLE PREPARATION ______

12 You will now make up the protein samples which you will use in Experiments 2 and 3. You will again be assigned the protein you used in Workshop I (Intro. to Protein Databases). Before weighing out your protein, you must have read ahead in the lab script and calculated the amounts of these substances you will need.

You will be making up your protein sample to be approximately 1 mg/mL by preparing a sample of known weight and volume. However, this will not be an accurate guide to concentration, as your protein may not be pure from the manufacturer and may be contaminated with salts or nucleic acids. For this reason, the protein sample you make in this section will be referred to as US, for sample of unknown concentration.

As mentioned above, you need to determine how much stock solution you will need to do the required protein concentration determination experiments outlined in Experiment 2 as well as acquire a circular dichroism spectrum of your protein in Experiment 3 (200 L of 0.1 mg/mL and 60 L of 1 mg/mL). Please fill in Table 2 below with the required volumes of 1 mg/mL stock needed for each assay and get this checked by a demonstrator in order to obtain your protein.

Table 2: Experiment Assay Vol ~ 1 mg/mL US Vol 1 mg/mL SS II A B C D III  Total Volume Total Mass 

Once approved, you will be given the protein and will make up this solution in your buffer solution. 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).

QUESTIONS (ANSWERS TO BE SUBMITTED IN LAB REPORT) ______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?

13 14 EXPERIMENT II: 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 II. The first is the protein stock solution you made in Experiment I, 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 (exactly 1 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 or on graph paper, but one of these plots 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.

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 using a Perkin Elmer Lambda 25 spectrometer. You will then use this information, along with the extinction coefficient determined in the data base exercise, to calculate concentration.

15 Protocol. To collect a baseline, put ~ 2 mL 18.2 M water in either a clean dry or clean water rinsed quartz absorbance cuvette. Set the instrument parameters as follows:

Scan Data interval 0.1 nm Start wavelength 400 nm Stop wavlength 200 nm

Instrument Scan speed 240nm/min Lamp UV On Lamp VIS On Lamp change 326 nm

Sample Number of samples 1

Run a baseline/background spectrum with the cuvette in the sample position.

To obtain the sample spectrum, 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 3 times). Dry cuvette either with nitrogen line (or air if nitrogen is not available). Pipette directly into the cuvette: 1 mL of your US; add 1 mL 18.2 M water. Measure a spectrum using the same parameters as the baseline. Save your data directly onto a memory stick as an ASCII file.

QUESTIONS (ANSWERS TO BE SUBMITTED IN LAB REPORT) ______1. 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 1 3 1 the rationale behind this equation? (*cystine, max~120 mol dm cm ; Tyr, max ~ 1 3 1 1280 mol dm cm ; Trp, max~ 5690).

2. If lysozyme has a molecular weight of 14314, nW = 6, nY = 3, nC = 8, determine its . 1 3 1 Compare this with the experimental value of 280 = 37932 mol dm cm . The values for chymotrysinogen are: 25670, 8, 4, 10, 51340. Comment.

16 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.

H O R H O

N C C N C H

Cu(II)

C N C H C N

O H R O H

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; 0.25 mg protein; 0.5 mg protein; 1.0 mg protein; and 1.5 mg protein from the 1 mg / mL SS protein solution provided. Also prepare duplicates of 2 different concentrations of US that you predict will lie within the range defined by your calibration curve (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 37C. 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 37C. 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. Finally, back calculate the concentration of your original (~ 1 mg/ mL) stock.

17 * 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.

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 dye 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.)

You will be using the Biorad assay, which is a commercial version of the Bradford protein assay. The accuracy of this assay depends on accurate pipetting and above all thorough mixing. You will be using automatic pipettes make sure that you know how to use them before attempting the assay. There is a small error introduced into this method by the calibration points having slight differences in volume. This error is small and can be ignored.

Protocol. You will again begin by preparing SS standards for a calibration curve containing: 0 µg, 5 µg, 10 µg, 20 µg, 50 µg of protein from the 1 mg / mL SS protein solution provided. Also prepare duplicates of 2 different concentrations of US. Place required volumes, x µL, of the SS and US solutions in clean, dry test tubes. Add 3 mL of the diluted Biorad reagent and mix thoroughly (could vortex (avoid excess foaming) or mix several times by gentle inversion of test tube). After a period of 10 minutes, determinine A595. Plot A595 versus the amount of

18 protein in each SS sample. Read unknowns from the standard curve, and determine their concentrations. Then back calculate the concentration of your original (~ 1 mg/ mL) stock.

ASSAY D: BCA METHOD ______

Once again, the SS solution will be used to prepare samples for a calibration curve. Table 3 below summarises the SS 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.

Table 3: Protein concentration Vol. 1 mg/mL SS solution Vol. buffer or water (g/mL) (L) (L) 1000 100 0 500 50 50 200 20 80 50 5 95 0 0 100

Protocol. A stock reaction mixture containing 20 mL BCA reagent, Pierce No. 23223 and 285 L 4%

CuSO4 has been prepared for you. For each analysis: make 100 L protein solution of the desired concentration in buffer or water. Mix well. Then add 2 mL of the reaction mixture. Mix well. Incubate at 37C 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, and as before back calculate the concentration of your original (~ 1 mg/ mL) stock.

Prepare a table containing the concentrations of your US stock solution as determined by Assays A, B, C, and D. Compare them and discuss reasons from differences.

19 EXPERIMENT III: 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). Dry it with air or a 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 II. Determine the -helical content of your protein as given below and compare the answer with that from the crystallographic data base.

-1 3 -1 IAMBEC: assume that 100% -helical protein has 208 nm ~ -12 mol dm cm . 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.

Ensure 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.