To analyze:
Which are the important features of proteins that determine their electrophoretic mobility? Based on your answer, describe how the substitution of single amino acid can alter the electrophoretic mobility of a protein.
Introduction:
Gel electrophoresis is the bioanalytical technique of separation of different proteins or
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GENETIC ANALYSIS: AN INTEG. APP. W/MAS
- Consider a protein in which a negatively charged glutamic acid side chain (pKa = 4.2) makes a salt bridge (ion-ion interaction) with a positively charged histidine side chain (pKa = 6.5). Part A Do you predict that this salt bridge will become stronger, become weaker, or be unaffected as pH increases from pH = 7.2 to pH = 7.8? The salt bridge will become stronger. The salt bridge will become weaker. The salt bridge will be unaffected. Submit Part B Previous Answers Correct At pH = 7.2 the glutamic acid (Glu) side chain will carry a charge of ~ -1 (at 3 pH units above the pKa for Glu, the side chain will be almost fully ionized); whereas the histidine (His) side chain will carry a charge of < +0.5 (at pH = pK₂ the charge on His would be +0.5; since pH = 7.2 is above its pKa, it will carry less (+) charge as it becomes more deprotonated). As the pH increase to 7.8, the charge on Glu will remain ~ -1 and the charge on His will decrease; thus, this salt bridge is predicted to become weaker…arrow_forwardA researcher isolates a new protein and finds that it contains 0.755% serine by weight upon amino acid analysis. The molecular mass of serine is 105 g.mol-¹. What is the minimum molecular mass for the protein assuming only one Ser residue per protein molecule? mmin = The researcher estimates the molecular mass of the protein using gel electrophoresis. From this estimated molecular mass, the researcher concludes the protein contains 3 Ser residues. What is the estimated molecular mass of the protein as shown by gel electrophoresis? g.mol-¹ mest = g.mol-1arrow_forwardShow the approximate position of the indicated four amino acids following electrophoresis. Here you have to consider both the polarity and the value of the total net charge of the amino acid. Assume that an amino acid with a net charge of +2 will migrate twice as far towards the minus pole as an amino acid with a net charge of +1. To provide some guidance on how to indicate the amino acids, we show in the image the migration of an example amino acid with unknown net charge; you do not have to consider its migration behaviour in your decision to place the four amino acids.arrow_forward
- What is the optimum pH to separate a mixture of lysine, arginine, and cysteine using electrophoresis? Draw the structures of the three amino acids in the protonation state that would predominate at the pH you have chosen. For each amino acid, indicate the net charge at the chosen pH as well as the direction of migration and relative mobility in the electric field.arrow_forwardConsider the following peptide to answer the questions below: A M A K K E S H I F T A I D E What is the total charge of this peptide at pH = 5? What is the N terminal amino acid (full name)? C terminal amino acid? How many fragments would result if this peptide underwent proteolytic cleavage with V-8 protease and what are these fragments (list them)? Would this peptide be a candidate to form an alpha helix? Why or why notarrow_forwardYou are given an equimolar (0.10 mM) mixture of Ubiquitin protein that is free/pure of any other macromolecules (e.g. nucleic acids) in a pH 7.0 phosphate buffer How can you purify Ubiquitin using Ion-Exchange chromatography (IEC)? Propose a plan using steps to purify this protein. Basic rules: Affinity chromatography (e.g. His Tag purification) is not an allowed step because these proteins are in their native state (i.e. – do not have a polyhistidine tag). Also, you are not allowed to rely solely on the color of certain proteins (e.g. cytochrome C and GFP) in your characterization/proposal. You are allowed to buffer exchange (i.e. – switch buffers) but try to keep your pH’s in a reasonable range (5.5 – 8.5) or you risk denaturing your protein. Assume proteins with the same charge (positive or negative) are not easily separated using IEC. Assume all proteins can be resolved/visualized on an SDS-PAGE gel. Ubiquitin Molecular weight (8663.02); pI (6.56); ε (M-1 cm-1) ignoring C’s…arrow_forward
- By means of an annotated flow diagram, show what information is used to determine the conformation or three-dimensional structure of a protein molecule.arrow_forwardWhat is binding energy? What do negative and less negative energies represent? How does this relate to protein function?arrow_forwardYou are working on an enzyme that binds to a steroid hormone. Answer the following questions: • Is this likely to be a globular protein? Explain your answer. • The substrate is a steroid hormone. How does the enzyme bind to the steroid? (think about the non-covalent interactions that allow this to occur) • Describe the difference in net charge of the surface of this protein compared to the core of the protein. Are they most likely to be different or the same? Explain your answer. • Since this enzyme is extracellular, where in the cell would this enzyme be translated?arrow_forward
- What is the optimum pH to separate a mixture of lysine, arginine, andcysteine using electrophoresis? Draw the structures of the three amino acidsin the protonation state that would predominate at the pH you have chosen.(as shown) For each amino acid, indicate the net charge at the chosen pH as well as the direction of migration and relative mobility in the electric field.arrow_forwardImagine the main chain of a protein bends back on itself, so that two amino acid residues R, and R, come close to each other. In the table below are four possibilities for what R, and R, might be. In each case, decide whether a specific interaction could form between the residues. If a specific interaction could form, give the name of the interaction. R₁ R₂ threonine cysteine glutamine arginine cysteine tyrosine phenylalanine glutamate specific interaction? Oyes no yes O no O yes O no Oyes O no name of specific interaction 0 0 0 0 Xarrow_forwardImagine the main chain of a protein bends back on itself, so that two amino acid residues R, and R, come close to each other. In the table below are four possibilities for what R, and R, might be. In each case, decide whether a specific interaction could form between the residues. If a specific interaction could form, give the name of the interaction. R1 R2 specific interaction? name of specific interaction O yes serine tyrosine O no О yes valine threonine O no O yes threonine glutamine O no О yes cysteine cysteine noarrow_forward
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