Unit 2 1 UNIT 2 PART A: PROTEIN STRUCTURE PART B: PROTEIN PURIFICATION AND ANALYSIS PART A: LEVELS OF STRUCTURE IN PROTEIN ARCHITECTURE Asignment: Nelson & Cox, review pp. 43 - 51 (stop at "Solutes afect.."), 84 - 85, 92 - 93, 113 - 123, 129 - 131, 135 - 138, 140 - 148. The biological activity of a protein depends on its thre-dimensional structure and its interaction with other molecules. Detailed knowledge of protein structure contributes greatly to our understanding of exactly how a protein functions. We have learned in Unit 1 that proteins are made up of linear polymers of amino acids. In this unit we wil learn how the amino acid sequence dictates the thre dimensional structure of proteins and how hydrophobic interactions and secondary structures are involved in the folding of proteins to produce the tertiary structure. 1. What is a prosthetic group (p. 84)? 2. There are 20 common amino acids found in proteins. Many of these amino acids can be modified after synthesis of the polypeptide chain. List several of these modification types. (Table 3-4, pp. 85). Unit 2 2 3. Levels of Structure in Proteins (p. 92). Distinguish betwen each of the four levels of protein structure. For secondary structure, a beter definition states simply that the secondary structure of a protein consists of regularly repeating conformations of the polypeptide backbone such as ? helices and ? pleated sheets. These always involve hydrogen (H) -bonds in a regular patern. A more complete definition of the tertiary structure of a globular protein is that it consists of the completely folded, 3-dimensional, biologicaly active (or native) conformation of a single polypeptide protein. In tertiary structure the nature of the amino acid side chains is an important factor. 4. Atomic forces, interactions, or bonds Proteins are held together and al interactions betwen molecules are caused by hydrogen bonds, ionic bonds or interactions, hydrophobic interactions, and van der Wals interactions. Discuss these weak non-covalent interactions that stabilize a protein's conformation (review pp. 43 - 51, pp. 114 - 115). 5. Polypeptide chains are flexible but the peptide bond does not rotate. Refering to Fig. 4-2 (p. 116), explain the following: a. The peptide unit is rigid and planar: the N and C atoms of each peptide bond and the atoms atached to them al lie in one plane. b. There is no fredom of rotation about the bond betwen the carbonyl carbon atom and the nitrogen atom of the peptide unit. c. The hydrogen of the amino group is nearly always trans (opposite) to the oxygen of the carbonyl group because the cis form is stericaly hindered. d. The link betwen the ?-carbon atom and the carbonyl carbon atom is a pure single bond. The bond betwen the ?-carbon atom and the peptide nitrogen atom is also a pure single bond. Thus there is a large degre of rotational fredom about each of these bonds. But there are restrictions as shown by the Ramachandran plot (Fig. 4-3, p. 117). What is the basis of this restriction? Unit 2 3 6. Secondary structure: the ? helix (pp. 117-120) a. The ? helix found in proteins is right-handed (Box 4-1, p. 118). Hold your right hand with the thumb up and fingers curved behind. Place this hand next to the helix and you wil note that your fingers wil naturaly follow the ? helix upward. This won't work with your left hand. b. Amino acid side chains and hydrogen atoms on each ?-carbon project out from the axis of the ? helix. The atomic radii are such that there is no significant hole in the middle. c. Intramolecular hydrogen bonds connect the carbonyl oxygen of one amino acid residue to the peptide hydrogen in the fourth succeding amino acid residue. d. The hydrogen bonds are nearly paralel to the axis of the helix. e. There are 3.6 amino acid residues per turn of the ? helix. f. The ? helical structure is esentialy rigid and rodlike. 7. Secondary structure: the ? pleated sheet (pp. 120 - 123). The ? sheet is built from a combination of several regions of the polypeptide chain, in contrast to the ? helix which is built from one continuous region. These regions are generaly 5 to 10 residues long. The polypeptide chains run adjacent to each other and hydrogen bonds can form betwen C=O groups of one ? strand and NH groups on an adjacent ? strand. The ? sheets that are formed from several such ? strands are "pleated". The C ? atoms are a litle above and below the plane of the sheet and the side chains (residues) point above and below the sheet. a. Given the following structure, draw an arow above each chain to indicate the N ? C? ? C direction (caled N ? C). Label the paralel and anti-paralel chains. Unit 2 4 b. Notice that, as in the ? helix, al four atoms atached to the peptide N and C lie in the same plane. c. Note that in contrast to the ? helix, the ? pleated sheet is stabilized by hydrogen bonds betwen (N-H) and (C=O) groups of chain(s) that run side by side. 8. Secondary structure: loops and ?-turns (p. 121). A combination of secondary structures, ? helices and ? sheets form the stable hydrophobic core of the protein molecule. For a polypeptide chain to abruptly reverse its direction it needs to leave the hydrophobic core. Turns are formed at the surface of the protein molecule. These regions are rich in charged, polar, hydrophilic residues. The main chain C=O and NH groups of these loop regions, are exposed to the aqueous solvent and can form H- bonds with water molecules. In this course we wil use the term, loop, to describe a turn that connects either two ?-helices or a turn connecting a ?- strand to an ?-helix. ?-turns are a type of loop that specificaly connects two ?-strands. Unit 2 5 9. Supersecondary structure: The ? ? ? unit (Fig. 4-17, p. 131); a ? strand separated from a paralel ? strand by an ? helix. Draw either ? helices or ??turns connecting the appropriate polypeptide segments shown in the structure in objective 7a to form a sheet made from a single polypeptide chain. Note: There are many possible answers. 10. For a review of secondary and supersecondary structure, run through the 3-D Structure activity entitled "Protein Architecture" on the Lehninger Principles of Biochemistry 5th ed. website. These activities are browser specific. For details, read the notes on the external link from the Blackboard website (subsection coursework related links by unit, Unit 2). 11. Tertiary Structure. a. Distinguish betwen a domain (p. 135) and a subunit (p. 123). Note: With al the sequence information now available, the identification of domains with known function is an important aspect of gene analysis. Many polypeptides consist of a string of domains and if the function of these domains are known, the probable function of the protein being studied can be hypothesized. Unit 2 6 b. Side chain hydrogen bonds are important in the stabilization of tertiary structure. 1) Given the following table of H-bonding groups in proteins, group them acording to their hydrogen-bonding potentialities (hydrogen donors, hydrogen aceptors). 2) Are the hydrogen-bonding modes of the ionizable residues pH-dependent? H - Bonding in Proteins c. What other types of interactions are involved in stabilization of the native tertiary structure (pp. 114 - 115)? Unit 2 7 d. Use myoglobin as an example of a protein that is built primarily of ? helices. Where are hydrophobic and hydrophilic side chains located in the myoglobin molecule (Fig. 4-15, p. 130)? 12. Protein Folding (pp. 140 - 148) In the conversion of an unfolded polypeptide chain into a folded protein many possible conformations are searched to find the energeticaly most favorable form. The presence of non-polar molecules in water limits the number of diferent ways in which the surrounding water molecules can hydrogen bond to each other. If the exposure of non-polar groups to water is minimized, then an energeticaly favorable state is reached. This tendency to force non-polar groups out of water is caled the hydrophobic efect. In the initial stages of protein folding in aqueous solution, the non-polar side chains of the unfolded protein are exposed to water and therefore are driven to asociate by short-range hydrophobic interactions. The hydrophobic side chains collect in the interior of the protein and this proces provides a significant part of the driving force for folding. It has now been established that, simultaneously with the hydrophobic collapse, stable secondary structures necesary for the formation of the complete tertiary structure, are formed. These secondary structures stabilize the polar peptide bond in the hydrophobic core of the protein. a. Discuss the evidence that the amino acid sequence of ribonuclease specifies its thre-dimensional structure. In doing so, consider the following (pp. 140 - 142; Fig 4-26, p. 141): Unit 2 8 1) The four disulfide bonds in ribonuclease cannot be readily reduced by ?-mercaptoethanol unles the protein is partly unfolded by agents such as urea or guanidine hydrochloride. What is the mechanism of action of the two reagents, do they disrupt covalent or noncovalent interactions? 2) Describe the conformation of ribonuclease that has been treated with urea and ?-mercaptoethanol. What is the meaning of the phrase "to denature a protein"? 3) How can the denatured and reduced ribonuclease be converted to its native form? 4) Why did the ?Anfinsen? experiment prove that the primary sequence specifies the native structure? Note: Very diferent amino acid sequences can generate strikingly similar protein folds. For example 226 diferent globins with only two fully conserved residues al have the same main-chain conformation. b. Models of protein folding: 1) Distinguish betwen the two models presented for protein folding (Figs. 4-27 and 4-28; pp. 142 - 143). Note that although most proteins fold by a proces that incorporates features of both models, most biochemists consider the hydrophobic collapse to be the most important interaction that drives a globular protein to fold. As this occurs, secondary structures are formed in order to stabilize the polar backbone in the hydrophobic core of the protein and molten globule. 2) Use Fig. 4-28 (p. 143) to describe the thermodynamics of protein folding. Define the term "molten globule". 3) Although it is usualy asumed that a protein has one lowest energy structure, it is now realized that many proteins can have diferent structures depending on what they are bound to and in some cases regions of proteins are normaly in a random coil (or unstructured) state. Unit 2 9 c. Discuss the role of molecular chaperones and chaperonins in protein folding (pp. 143 - 144, you do not need to worry about the details of the mechanisms!). 1) Most proteins that contain disulfide bonds are extracelular. In living cels, discuss the role of protein disulfide isomerase (PDI; which is located in the lumen of the endoplasmic reticulum) , on protein folding (p. 144). 2) Discuss the role of peptide prolyl cis-trans isomerase (PI) in protein folding (p. 144). PART B: PROTEIN PURIFICATION AND ANALYSIS Asignment: Nelson & Cox, pp. 76 (Fig. 3-6 and Box 3-1), 85 - 100, 132 - 134 (Box 4-5), 173 - 174. 1. Discuss each of the following terms as it relates to protein purification (pp. 85 - 88): a. Crude Extract b. Diferential & sucrose density centrifugation (Se Fig. 1-8, p. 7) c. Fractionation d. Dialysis e. Column Chromatography Chromatography is generaly one of the most important steps in a protein purification scheme. 1) Describe ion exchange chromatography (p. 86). a) On what basis are proteins separated using this technique? b) What is the overal charge on a protein when the pH < pI? pH = pI? pH > pI? Unit 2 10 c) Since the charge on a protein varies as a function of pH, a protein bound to an ion exchange column can be eluted by shifting the pH so that it becomes uncharged - or asumes the opposite charge. Name another method for eluting bound proteins. 2) Describe how two proteins of diferent molecular weight can be separated from each other by size-exclusion chromatography ("gel filtration" chromatography"; pp. 87 - 88). a) Absorbance of UV light at 280nm is often used as a crude measure of protein concentration. Why do protein solutions absorb light at 280nm (Fig. 3-6 (p. 76) and Box 3-1 (p. 76))? Note there are other methods of protein quantitation that are more sensitive and more specific than A 280 . An example is the dye binding asay devised by Bradford et al. The dye used in this asay, coomasie blue, is the same dye that is used to stain SDS- PAGE gels alowing visualization of protein bands. You wil se examples of stained gels later in this unit. Unit 2 11 b) The diagram that follows is an "elution profile" for a size-exclusion chromatography column. Does Protein A or Protein B have the higher molecular weight? (Note that as a column runs, the liquid that flows through the column is collected into a series of test tubes, or fractions. In the figure below, fraction 1 on the left is the first liquid to emerge from the column and the fractions on the right emerged much later after a larger volume of liquid was run through the column.) A B fraction number A b s o r b a n c e ( 2 8 0 n m ) 3) Describe the separation of proteins by affinity chromatography (p. 88). (Note that the binding groups joined to the resin can be substrate-like molecules, antibodies, etc. Although this is an extremely powerful technique, appropriate resins are only available for a very smal number of proteins! This technique has become a standard method in the purification of proteins encoded by recombinant DNA. In these cases, the DNA is modified so that the encoded protein wil contain a chain of histidine residues. The nickel afinity resin binds histidine giving a shortcut for the purification of the protein.) 4) What does HPLC stand for (p. 88)? 2. Proteins can be characterized by electrophoresis (pp. 88 - 91). Unit 2 12 a. Use Fig. 3-18 (p. 89) to describe the separation of proteins by SDS gel electrophoresis. 1) Look at the structure of the detergent SDS on p. 89. Point out the polar and nonpolar parts of the molecule. 2) Mixtures of SDS and protein are heated to denature the protein. When cooled, the SDS preserves the denatured form by hydrophobic interactions forming an elongated micele around the extended peptide backbone. (Note: the actual structure is not known). What is the approximate ratio of SDS to the number of amino acids (p. 89)? Describe how the charge on the complex compares with the original charge on the protein. 3) In the presence of SDS, what property of the protein determines the rate of migration? 4) Wil smal protein molecules move faster or slower than large ones? Explain. 5) Use Fig. 3-18b (p. 89) to describe how an SDS gel can be used to determine the purity of a protein sample and its subunit composition. SDS gels are also used to determine a protein's size. 6) Use Box 1-1 (p. 14) to distinguish betwen molecular weight and molecular mas. What units are used for each measurement? Use Fig, 3-19 (p. 90) to determine the molecular weight of the unknown protein. Be sure to expres your answer using appropriate units. b. What does pI stand for (p. 90)? Use Fig. 3-20 (p. 90) to describe isoelectric focusing. Unit 2 13 c. Figure 3-21 (p. 91) describes two-dimensional electrophoresis. 1) How does a protein ?spot? on the left side of the gel difer from a protein ?spot? on the right side? 2) How does a ?spot? on the top of the gel difer from a ?spot? on the bottom? 3. Distinguish "activity" from "specific activity" (p. 91). Use Table 3-5 (p. 88) to discuss the results of a protein purification in terms of total protein, total activity, and specific activity. 4. Run through the Flash Protein Purification Tutorial . Aces the link using the external links (Coursework related links by unit) section of the course Blackboard web site. Carefully follow the instructions at the blackboard external link to determine which browser to use! 5. Without going into the details of the chemistry of the Edman degradation, outline the strategy behind this technique (p. 95). For technical reasons, it is usualy only possible to determine the sequence of approximately 10-50 amino acids using the Edman degradation. If a polypeptide is broken into smaler overlapping fragments then each fragment can be sequenced and the total amino acid sequence determined. 6. Remember from Unit 2A that some amino acids are modified. Electrospray Mas Spectroscopy is a method alowing identification of such modifications in a protein. Using general terms, describe Electrospray Mas Spectroscopy (Box 3-2, pp. 98 - 100)? 7. Antibody techniques (pp. 173 - 174) a. Distinguish betwen polyclonal and monoclonal antibodies (p. 173). b. What is ELISA and what is it used for (p. 174)? Use Fig. 5-26 (p. 174) to describe the key steps in the procedure. Describe how ELISA could be used to test for the presence of antibodies against HIV virus in an infected individual. Unit 2 14 c. What is the purpose of an imunoblot asay (p. 174)? 1) How is an imunobot prepared? 2) In Fig. 5-26c (p. 174), explain why only a single band is visible in each lane of the imunoblot while several are visible in the SDS gel. Note: What Nelson & Cox cal an imunoblot asay is usualy caled a estern blot. 8. X-ray crystalography and 2D NMR are used to determine the structure of proteins. a. In general terms, discuss the key steps in x-ray crystalography. (Box 4-5, pp. 132 - 133). b. The esence of how NMR is described in Box 4-5 (pp. 133 - 134). NMR provides information on the distance betwen protons in a structure and if enough distances are determined, a model can be built. In this course, you wil not be responsible for the details of NMR but you should realize that it has the tremendous advantage of not requiring crystal preparation. NMR also gives a beter picture of the protein in the dynamic state. Jim Blankenship Microsoft Word - U02_F08.doc
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