Fluid Mechanics With Engineering Applications by E. John Finnemore and Joseph B Franzini. 10th edition
Mechanics of Materials CH05 Probs62-69Full description
Chapter 5: Proteins: Primary Structure
Matching A) B) C) D) E) F) ) !) ") $) &) () ) N) *) +) ,
electropho phoresi esis hydr hydrop opho hobi bicc inte interracti action on enzy enzyme me-l -lin inke ked d immu immuno noso sorb rbent ent assay assay thre threee-di dime mens nsio iona nall shap shapee N-terminal am amino acid negative ch charge nucleases chromophore #oaming high le level e% e%pression '-merca rcaptoetha ethan nol positive charge cation e%change nge p I chymotrypsin C-term erminal amino ac acid odium do dodecyl su sul#ate
./ *ne o# the reasons the primary structure is important important #or a protein is that it determines the 00three-dimentional shape0000 the molecule adopts in a1ueous solutions/ Ans2 D (evel o# Di##iculty2 Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
'/ "# the cDNA #or a protein has been cloned5 it may be possible to obtain large 1uantities o# the protein by 00high level e%pression000000000000000 in bacteria/ Ans2 $ (evel o# Di##iculty2 Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
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6/ 7o help prevent denaturation o# proteins in in solution5 steps are taken to avoid 00#oamin0000000 and adsorption to sur#aces/ Ans2 " (evel o# Di##iculty2 Di##iculty2 oderate ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
8/ olecules that contain a9n) 00chromophor0000 are capable o# absorbing light/ light/ Ans2 ! (evel o# Di##iculty2 Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
3/ "# antibodies to the protein protein being assayed are available5 a9n) 000000 can be carried out/ Ans2 C (evel o# Di##iculty2 Di##iculty2 oderate ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
:/ "n general5 proteins are least least soluble in ;ater ;hen the p! is close to the the 0p"00000/ Ans2 N (evel o# Di##iculty2 Di##iculty2 oderate ection2 3/'/B (earning ob4ective2 Protein Purification and Analysis
000hydrophoic interaction000 chromatography is a method o# #ractionating #ractionating a protein mi%ture mi%ture according to di##erences in polarity/ Ans2 B (evel o# Di##iculty2 Di##iculty2 Easy ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
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=/ "n order #or DEAE to act as an anion e%changer5 it must have a 00positive0000/ Ans2 ( (evel o# Di##iculty2 Easy ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
>/ "n 000cation e%change000 chromatography5 a protein mi%ture must be applied to the column at a lo; p! so that the proteins ;ill have a net positive charge and bind to the column/ Ans2 (evel o# Di##iculty2 oderate ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
.?/ "n D-+AE5 disul#ide-linked polypeptides can be separated a#ter reacting the protein #irst ;ith 0 &) '-mercaptoethanol 00000/ Ans2 & (evel o# Di##iculty2 Easy ection2 3/'/D (earning ob4ective2 Protein Purification and Analysis
../ Either dansyl chloride or Edman@s reagent can be used to identi#y the 0N-terminal00000 o# a protein/ Ans2 E (evel o# Di##iculty2 oderate ection2 3/6/A (earning ob4ective2 Protein Sequencing
.'/ 7he endoprotease chyrotrpsin000000 cleaves polypeptides on the C-terminal side o# certain bulky hydrophobic amino acid residues/ Ans2 * (evel o# Di##iculty2 oderate ection2 3/6/B (earning ob4ective2 Protein Sequencing
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Multiple Choice .6/ +roteins are synthesized in vivo by the translation o# A) cDNA/ B) tNA/ C) rNA/ D) e%ons/ E) mNA/ Ans2 E (evel o# Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
.8/ ince there are '? standard amino acids5 the number o# possible linear polypeptides o# length N can be e%pressed as2 A) n × 20 B) '?n C) '? × .?n D) n'? E) n × .?'? Ans2 B (evel o# Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
.3/ Natural proteins most commonly contain linear polypeptides bet;een .?? and .??? residues in length/ *ne o# the reasons polypeptides outside this range may be dis#avored is that A) larger polypeptides ;ould likely be insoluble/ B) smaller polypeptides do not #orm stable #olded structures/ C) smaller polypeptides typically assemble into prion-like aggregates/ D) amide linkages are not strong enough to keep larger polypeptides intact/ E) ribosomes are unable to synthesize larger polypeptides/ Ans2 B (evel o# Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
8
.:/ 7he vast ma4ority o# polypeptides contain bet;een 000000 amino acid residues/ A) .? and 3? B) 3? and .?? C) .?? and .??? D) .??? and '??? E) '??? and 685??? Ans2 C (evel o# Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
. hich o# the #ollo;ing has the most dramatic in#luence on the characteristics o# an individual protein A) the amino-acid se1uence B) the amino-acid composition C) the location o# its encoding gene ;ithin the genome D) the stereochemistry at the α-carbon E) the se1uence o# tNA molecules involved in its translation Ans2 A (evel o# Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
.=/ hich statement about insulin is correct A) "nsulin is composed o# t;o polypeptides5 the A chain and the B chain/ B) "nsulin contains an intrachain disul#ide bond/ C) "nsulin contains interchain disul#ide bonds/ D) 7he A chain and the B chain o# insulin are encoded by a single gene/ E) All o# the above are correct/ Ans2 E (evel o# Di##iculty2 Easy ection2 3/. (earning ob4ective2 Polypeptide Diversity
3
.>/ A #ast and common method #or determining the protein concentration in column e##luent is A) tandem mass spectrometry/ B) salting in ;ith ammonium sul#ate/ C) drying a portion and ;eighing the solid/ D) measuring light absorption at '=? nm/ E) Edman degradation/ Ans2 D (evel o# Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
'?/ 7he salting in o# proteins can be e%plained by2 A) salt counter-ions reducing electrostatic attractions bet;een protein molecules/ B) salt ions reducing the polarity o# the solution/ C) salt ions increasing the hydrophobic interactions/ D) releasing hydrophobic proteins #rom nonpolar tissue environments/ E) hydration o# the salt ions reducing solubility o# proteins/ Ans2 A (evel o# Di##iculty2 oderate ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
'./ 7he 1uantitation o# proteins due to their absorbance at '=? nm 9 region) is due to the large absorbtivity o# the 00000000 amino acids/ A) anionic B) dansylated C) cleaved D) polar E) aromatic Ans2 E (evel o# Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
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''/ hich o# the #ollo;ing GassaysH ;ould be most speci#ic #or a particular protein A) Brad#ord assay B) absorptivity C) radioimmunoassay D) molar absorptivity E) amino acid analysis Ans2 C (evel o# Di##iculty2 oderate ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
'6/ An enzyme-linked immunosorbent assay re1uires A) a radioactive substrate/ B) a radioactive standard #or binding to the antibody/ C) aromatic amino acids/ D) an antibody that binds the protein o# interest/ E) a catalytic antibody/ Ans2 D (evel o# Di##iculty2 oderate ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
'8/ E("A is an e%ample o# a9n)2 A) enzyme assay/ B) biological assay/ C) binding assay/ D) immunological assay/ E) none o# the above Ans2 D (evel o# Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
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'3/ Iou are puri#ying a nuclease by a##inity chromatography/ 7o determine ;hich #ractions contain the protein o# interest5 you test samples o# all #ractions #or their ab ility to break do;n DNA/ 7his is an e%ample o# A) a binding assay/ B) a biological assay/ C) an enzyme assay/ D) an immunological assay/ E) none o# the above Ans2 C (evel o# Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
':/ A radioimmunoassay re1uires A) an enzyme-linked antibody/ B) a coupled enzymatic reaction/ C) a radiolabeled antibody/ D) a catalytic antibody/ E) a radiolabeled standard protein that is used to compete #or binding to the antibody/ Ans2 E (evel o# Di##iculty2 oderate ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
' Five graduate students prepare e%tracts #rom 3 di##erent tissues/ Each student measures the total amount o# alcohol dehydrogenase and the total amount o# protein in his or her e%tract/ hich e%tract has the highest speci#ic activity 7otal protein 9mg)
Ans2 B (evel o# Di##iculty2 Easy ection2 3/'/A (earning ob4ective2 Protein Purification and Analysis
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'=/ hich physical characteristic is not commonly used in protein separation A) solubility B) stereochemistry C) size D) charge E) polarity Ans2 B (evel o# Di##iculty2 Easy ection2 3/'/B (earning ob4ective2 Protein Purification and Analysis
'>/ Adding additional salt to a protein solution can cause2 A) an increase in solubility called Gsalting inH/ B) a decrease in solubility called Gsalting outH/ C) protein precipitation #rom solution/ D) all o# the above E) none o# the above Ans2 D (evel o# Di##iculty2 Easy ection2 3/'/B (earning ob4ective2 Protein Purification and Analysis
6?/ A #irst step in puri#ying a protein that ;as initially associated ;ith #atty substances ;ould be A) Coomassie Brilliant Blue dye staining/ B) analytical ultracentri#ugation/ C) E("A/ D) estern blotting/ E) hydrophobic interaction chromatography/ Ans2 E (evel o# Di##iculty2 oderate ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
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6./ 7he acronym !+(C stands #or A) hydrophobic protein li1uid chromatography/ B) high per#ormance li1uid chromatography/ C) hydrophilic partition li1uid chromatography/ D) high priced li1uid chromatography/ E) hydrostatic process li1uid chromatography/ Ans2 B (evel o# Di##iculty2 Easy ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
6'/ A techni1ue that can be used to separate proteins based primarily on the presence o# non polar residues on their sur#ace is called A) ion-e%change chromatography/ B) gel #iltration chromatography/ C) a##inity chromatography/ D) gel electrophoresis/ E) hydrophobic interaction chromatography/ Ans2 E (evel o# Di##iculty2 Easy ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis 66/ A techni1ue that can be used to separate proteins based primarily on their p I is called A) ion-e%change chromatography/ B) gel #iltration chromatography/ C) a##inity chromatography/ D) isoelectric #ocusing/ E) hydrophobic interaction chromatography/ Ans2 D (evel o# Di##iculty2 Easy ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
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68/ hich o# the #ollo;ing amino acids ;ould be last to elute at p! =/? #rom an anion-e%change column A) lysine B) alanine C) glutamic acid D) asparagine E) glycine Ans2 C (evel o# Di##iculty2 oderate ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis 63/ hich o# the #ollo;ing amino acids ;ould be #irst to elute at p! =/? #rom an anione%change column A) lysine B) alanine C) glutamic acid D) asparagine E) glycine Ans2 A (evel o# Di##iculty2 oderate ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
6:/ 7he p K .5 p K '5 and p& o# the amino acid lysine are '/'5 >/.5 and .?/35 respectively/ 7he p K .5 p K '5 and p& o# the amino acid arginine are ./=5 >/?5 and .'/35 respectively/ A student at D ;ants to use ion e%change chromatography to separate lysine #rom arginine/ hat p! is likely to ;ork best #or this separation A) ./3 B) '/3 C) 3/3 D) 3 E) .?/3 Ans2 E (evel o# Di##iculty2 Di##icult ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
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6 7he p K .5 p K '5 and p& o# the amino acid histdine are ./=5 >/65 and :/?5 respectively/ 7he p K .5 p K '5 and p& o# the amino acid arginine are ./=5 >/?5 and .'/35 respectively/ Iou have a mi%ture o# histidine and arginine5 ho; ;ould you try to separate these t;o amino acids A) anion e%change chromatography at p! ' B) anion e%change chromatography at p! 8 C) cation e%change chromatography at p! ' D) cation e%change chromatography at p! 8 E) cation e%change chromatography at p! > Ans2 E (evel o# Di##iculty2 Di##icult ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
6=/ hat can be done to increase the rate at ;hich a protein o# interest moves do;n an ione%change chromatography column A) reduce the ion concentration in the eluant B) add a small amount o# a non-ionic detergents to the eluant C) change the p! o# the eluant D) add a protease inhibitor to the eluant E) reduce the temperature o# the eluant Ans2 C (evel o# Di##iculty2 oderate ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis 6>/ !ydrophobic interaction chromatography can be used to separate proteins based on di##erences in A) ionic charge/ B) solubility/ C) size/ D) polarity/ E) binding speci#icity/ Ans2 D (evel o# Di##iculty2 Easy ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
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8?/ Iou are trying to separate #ive proteins5 ;hich are listed belo;5 by gel #iltration chromatography/ hich o# the proteins ;ill elute #irst #rom the column A) cytochrome c 9.' kDa) B) NA polymerase 9>> kDa) C) glutamine synthetase 9:'. kDa) D) inter#eron-γ 968 kDa) E) hemoglobin 9:' kDa) Ans2 C (evel o# Di##iculty2 oderate ection2 3/'/C (earning ob4ective2 Protein Purification and Analysis
8./ D-+AE separates proteins primarily due to di##erences in A) isoelectric point/ B) mass/ C) polarity/ D) solubility/ E) amino acid se1uence/ Ans2 B (evel o# Di##iculty2 Easy ection2 3/'/D (earning ob4ective2 Protein Purification and Analysis 8'/ hich o# these techni1ues is used to separate proteins mainly based on mass A) polyacrylamide gel electrophoresis 9in the absence o# D) B) D-+AE C) isoelectric #ocusing D) immunoblotting E) estern blotting Ans2 B (evel o# Di##iculty2 Easy ection2 3/'/D (earning ob4ective2 Protein Purification and Analysis
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86/ hich o# these techni1ues uses antibodies to detect very small amounts o# speci#ic proteins #ollo;ing separation by D-+AE/ A) immunoblotting B) silverstaining C) Coomassie Brilliant Blue staining D) E("A E) "A Ans2 A (evel o# Di##iculty2 Easy ection2 3/'/D (earning ob4ective2 Protein Purification and Analysis
88/ Disul#ide bonds can be cleaved using A) iodoacetate/ B) dansyl chloride/ C) '-mercaptoethanol 9β-E)/ D) trypsin/ E) phenylisothiocyanate/ Ans2 C (evel o# Di##iculty2 Easy ection2 3/6/A (earning ob4ective2 Protein Sequencing
83/ hich o# these reagents is commonly used to determine the number o# polypeptides in a protein A) iodoacetate B) dansyl chloride C) '-mercaptoethanol 9β-E) D) cyanogen bromide E) DEAE Ans2 B (evel o# Di##iculty2 Easy ection2 3/6/A (earning ob4ective2 Protein Sequencing
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8:/ Enzymes that hydrolyze the internal peptide bonds 9not the peptide bonds o# the terminal amino acids) o# a protein are classi#ied as A) o%idoreductases/ B) lyases/ C) endopeptidases/ D) nucleases/ E) e%opeptidases/ Ans2 C (evel o# Di##iculty2 Easy ection2 3/6/B (earning ob4ective2 Protein Sequencing
8 hich o# the #ollo;ing substances cannot be used to cleave peptide bonds in polypeptides A) trypsin B) cyanogen bromide C) endopeptidases D) '-mercaptoethanol E) pepsin Ans2 D (evel o# Di##iculty2 oderate ection2 3/6/B (earning ob4ective2 Protein Sequencing
8=/ hich o# these are commonly used to cleave peptide bonds in polypeptides A) '-mercaptoethanol 9β-E) B) dansyl chloride C) iodoacetate D) sodium dodecyl sul#ate E) trypsin Ans2 E (evel o# Di##iculty2 oderate ection2 3/6/B (earning ob4ective2 Protein Sequencing
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8>/ 7he peptide (eu ─ CysJArgJerJlnJet is sub4ected to Edman degradation/ "n the #irst cycle the peptide #irst reacts ;ith phenylisothiocyanate under basic conditions/ 7he product o# this reaction is incubated ;ith anhydrous tri#luoroacetic acid and subse1u ently ;ith an a1ueous acid/ hat are the products generated in the #irst cycle/ A) B) C) D) E)
+7!J(eu5 +7!JCys5 +7!JArg5 +7!Jer5 +7!Jln5 and +7!Jet +7!J(euJCysJArgJerJlnJet +7!Jet and (euJCysJArgJerJlnJet +7!J(euJCys and +7!JArgJerJlnJet +7!J(eu and CysJArgJerJlnJet
Ans2 E (evel o# Di##iculty2 oderate ection2 3/6/C (earning ob4ective2 Protein Sequencing
3?/ Edman degradation can be used to A) identi#y the N-terminal amino acid o# a polypeptide/ B) identi#y the C-terminal amino acid o# a polypeptide/ C) separate the subunits o# a multi-subunit protein/ D) cleave a protein at speci#ic sites/ E) cleave disul#ide bonds ;ithin a protein so that the individual polypeptides can be separated/ Ans2 A (evel o# Di##iculty2 Easy ection2 3/6/C (earning ob4ective2 Protein Sequencing
3./ Although a proteinHs primary se1uence can be in#erred #rom the nucleotide se1uence5 modi#ications such as 000000 can be determined most easily by tandem mass spectrometry #ollo;ed by protein database searching/ A) phosphorylation B) disul#ide crosslinks C) glycosylation D) acetylation E) all o# the above Ans2 E (evel o# Di##iculty2 Easy ection2 3/6/D (earning ob4ective2 Protein Sequencing
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3'/ 7he positive charge on proteins in electrospray ionization mass spectrometry is the result o# A) protons #ired at the gas-phase protein molecules/ B) protonated side chains o# Asp and lu residues/ C) protonated side chains o# Arg and (ys residues/ D) a high p!/ E) electrons #ired at the gas-phase protein molecules/ Ans2 C (evel o# Di##iculty2 oderate ection2 3/6/D (earning ob4ective2 Protein Sequencing
36/ 00000000000000 has emerged as a techni1ue #or protein se1uencing/ A) N spectroscopy B) ass spectrometry C) el electrophoresis D) +hylogenetic analysis E) (imited proteolysis Ans2 B (evel o# Di##iculty2 Easy ection2 3/6/D (earning ob4ective2 Protein Sequencing
38/ +rotein se1uences are customarily GreconstructedH #rom se1uenced #ragments because A) protein puri#ication invariably results in the #ragmentation o# the protein o# interest/ B) proteins are naturally and inevitably cleaved by proteolytic enzymes/ C) proteins are composed o# multiple subunits/ D) large polypeptides cannot be directly se1uenced/ E) all o# the above Ans2 D (evel o# Di##iculty2 Easy ection2 3/6/E (earning ob4ective2 Protein Sequencing
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33/ Iou have puri#ied a ne; peptide hormone/ 7o determine its amino acid se1uence you have digested the polypeptide ;ith trypsin and in a separate reaction you have cleaved the polypeptide ;ith cyanogen bromide/ Cleavage ;ith trypsin yielded 3 peptides that ;ere se1uenced by Edman degradation as sho;n in the #ollo;ing/ ./ '/ 6/ 8/ 3/
Determine the identity o# the N-terminal amino acid a#ter reconstructing the intact protein/ A) B) C) D) E)
Asp er !is lu "le
Ans2 A (evel o# Di##iculty2 Di##icult ection2 3/6/E (earning ob4ective2 Protein Sequencing
3:/ "n t;o homologous proteins5 ;hich residue is most likely to replace a lu residue as a conservative substitution A) Asp B) 7rp C) et D) "le E) (ys Ans2 A (evel o# Di##iculty2 Easy ection2 3/8/A (earning ob4ective2 Protein Evolution
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3 A phylogenetic tree depicts 00000000000 o# proteins/ A) #olding patterns B) hypervariable residues C) invariable residues D) evolutionary relationships E) gene se1uences Ans2 D (evel o# Di##iculty2 Easy ection2 3/8/A (earning ob4ective2 Protein Evolution
3=/ A protein that has had #e; changes in its amino acid se1uence over evolutionary history is labeled A) a #ibrinopeptide/ B) evolutionarily conserved/ C) random/ D) a product o# pseudogenes/ E) phylogenetic/ Ans2 B (evel o# Di##iculty2 Easy ection2 3/8/B (earning ob4ective2 Protein Evolution
3>/ +aralogous genes are A) genes that do not encode protein/ B) genes o# slo;ly evolving proteins/ C) relics o# genes that are not e%pressed/ D) genes o# rapidly evolving proteins/ E) the results o# gene duplication/ Ans2 E (evel o# Di##iculty2 oderate ection2 3/8/B (earning ob4ective2 Protein Evolution
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:?/ A #ast ;ay #or nature to generate ne; proteins is2 A) generation o# pseudogenes/ B) mutation by neutral dri#t/ C) shu##ling protein domains or moti#s/ D) hypervariable positions/ E) liberal substitution/ Ans2 C (evel o# Di##iculty2 Easy ection2 3/8/B (earning ob4ective2 Protein Evolution
:./ 00000000000 is an e%ample o# a very slo;ly evolving protein/ A) !istone !8 B) !emoglobin C) Cytochrome c D) Fibrinopeptides E) none o# the above Ans2 A (evel o# Di##iculty2 oderate ection2 3/8/B (earning ob4ective2 Protein Evolution
:'/ +roteins are o#ten constructed #rom multiple segments o# 8?-'?? amino acid residues5 commonly called A) pseudogenes/ B) hypervariable residues/ C) protolytic #ragments/ D) domains/ E) subunits/ Ans2 D (evel o# Di##iculty2 Easy ection2 3/8/B (earning ob4ective2 Protein Evolution
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SH!" A#S$E!
:6/ +roteins can vary in size #rom appro%imately 8? to 685??? amino acids/ a/ hy is there a lo;er limit to the size o# proteins b/ hy is there an upper limit to the size o# polypeptides Ans2 a/ +olypeptides shorter than 8? residues are unable to maintain a stable #old 9probably because there are not enough #unctional groups to help stabilize the #olded structure)/ b/ 7he longer the polypeptide5 the longer the corresponding mNA and gene5 resulting in an increase in mistakes made during transcription and translation/ Beyond a certain size every protein encoded #or by a very large gene ;ill include debilitating mistakes/ (evel o# Di##iculty2 oderate ection2 3/. (earning ob4ective2 Polypeptide Diversity
:8/ e are able to puri#y proteins because they di##er #rom each other in various physical or chemical properties/ (ist 3 physicochemical properties o# proteins that can be used as basis #or their separation/ ive a method o# separation based on each o# these properties 9match the method ;ith the property)/ Ans2 ./ solubility K salting outL '/ ionic charge K ion e%change chromatography5 electrophoresis 9in the absence o# D)5 or isoelectric #ocusingL 6/ polarity K hydrophobic interaction chromatographyL 8/ size 9mass) K gel #iltration chromatography 9size e%clusion chromatography) or D-+AE 3/ ability to bind to speci#ic molecules K a##inity chromatography (evel o# Di##iculty2 oderate ection2 3/'A (earning ob4ective2 Protein Purification and Analysis
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:3/ *ne techni1ue commonly used in protein puri#ication is chromatography/ a/ E%plain brie#ly the general principle o# column chromatography b/ Name #our types o# chromatography and indicate #or each o# these types the basis #or separation 9match types o# chromatography ;ith the properties that #orm the basis #or separation)/ Ans2 a/ "n chromatography there are t;o phases5 the stationary phase and the mobile phase/ 7he stationary phase is the column material and the mobile phase is the bu##er that runs through the column/ +roteins or molecules are separated based on their relative a##inities #or these t;o phases/ A molecule ;ith very high a##inity #or the stationary phase ;ill spend most o# its time associated ;ith the column material and ;ill move very slo;ly do;n the column/ A molecule ;ith high a##inity #or the mobile phase ;ill spend most o# its time suspended in the bu##er and ;ill move 1uickly through the column/ olecules ;ith high a##inity #or the stationary phase ;ill elute late #rom the column/ olecules ;ith a high a##inity #or the mobile phase ;ill elute early/ b/ ./ ion-e%change chromatography K ionic chargeL '/ gel #iltration chromatography K size 9mass)L 6/ hydrophobic interaction chromatography K polarityL 8/ a##inity chromatography K ability to bind to speci#ic molecules/ (evel o# Di##iculty2 oderate ection2 3/'C (earning ob4ective2 Protein Purification and Analysis
::/ "on e%change chromatography is a commonly used method #or separation o# biomolecules/ a/ hat are the t;o types o# ion e%change chromatography b/ "n ;hat order ;ould Arg5 al5 and lu elute #rom a carbo%ymethyl column at p! :/?/ Carbo%ymethyl is negatively charged at p! :/?/ c/ Iou are trying to puri#y a protein using ion e%change chromatography/ n#ortunately5 your protein remains bound to the ion e%change column or it is eluting very slo;ly/ hat are the t;o changes you can make5 to try to elute your protein more 1uickly #rom this column Ans2 a/ cation e%change chromatography and anion e%change chromatography/ b/ First lu5 then al5 #ollo;ed last by Arg5 because at p! :/? lu is has an electric charge o# -.5 al is neutral5 and Arg has an electric charge o# M./ c/ 7o make your protein move more 1uickly through the column you need to reduce the interactions ;ith the column material/ 7his can be done by changing the p! 9changing the charge on your protein) or by increasing the ion concentration in the eluant 9ions ;ill compete ;ith your protein #or binding to the column material)/ (evel o# Di##iculty2 oderate ection2 3/'C (earning ob4ective2 Protein Purification and Analysis
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: A variety o# chromatographic techni1ues are available #or protein puri#ication/ a/ E%plain brie#ly the principle o# hydrophobic interaction chromatography/ b/ Name three changes that can be made to the eluant that can be used to speed up elution o# the protein o# interest #rom a hydrophobic interaction chromatography column/ Ans2 a/ 7he column material is substituted ;ith nonpolar groups/ +roteins ;ith nonpolar regions on their sur#ace ;ill be driven to interact ;ith the column material as a result o# the hydrophobic a##ect/ b/ 7o ;eaken the interaction bet;een the column material and the nonpolar regions on your protein you can include detergents 9they ;ill shield nonpolar regions on proteins #rom interacting ;ith ;ater or other nonpolar groups)5 change the p! 9;ill change the polarity o# your protein)5 or reduce the ion concentration o# the eluant 9this ;ill make it less polar)/ (evel o# Di##iculty2 oderate ection2 3/'C (earning ob4ective2 Protein Purification and Analysis
:=/ Iou are interested in receptor protein-tyrosine kinases and you are puri#ying a novel phosphotyrosine-binding protein 9N+B+-.)/ "n order to characterize this protein you need to separate it #rom three other proteins 95 I5 and O) that are still present in your partially puri#ied material/ 7he proteins in your preparation have the #ollo;ing properties2
protein protein I protein O N+B+-.
binds to phosphotyrosine yes yes no yes
ize 9kDa) =3 '> 6. '=
p I ' '/> 8 3
a/ hat type o# separation techni1ue can be used to separate protein N+B+-. #rom protein b/ hat type o# separation techni1ue can be used to separate protein N+B+-. #rom protein I c/ hat type o# separation techni1ue can be used to separate protein N+B+-. #rom protein O Ans2 a/ *n a size 9mass) basis2 gel permeation chromatography5 D-+AE5 or ultracentri#ugation/ b/ *n a charge basis2 ion e%change chromatography or isoelectric #ocusing c/ *n a binding behavior basis2 phosphotyrosine a##inity chromatography (evel o# Di##iculty2 oderate ection2 3/'C (earning ob4ective2 Protein Purification and Analysis
'6
:>/ Iou have puri#ied the receptor #or a hormone by a##inity chromatography/ During gel #iltration chromatography under native conditions the receptor elutes bet;een pyruvate decarbo%ylase 9'3? kDa) and glutamine synthetase 9:'? kDa)/ During D-+AE5 in the absence o# reducing agents5 the receptor migrates as a single band o# appro%imately '6? kDa/ hen D-+AE is carried out in the presence o# '-mercaptoethanol the receptor migrates as t;o bands o# appro%imately >3 and .63 kDa/ E%plain this result/ Ans2 7he receptor is a heterotetramer composed o# t;o α subunits o# >3 kDa and t;o β subunits o# .63 kDa/ !eterodimers o# one α subunit and one β subunit are stabilized by disul#ide bonds/ 7;o heterodimers are held together by hydrophobic interactions/ "n the absence o# detergents during gel #iltration all subunits stay together and ;e observe a 8:? kDa protein/ During D+AE 9in the absence o# reducing agents) D inter#eres ;ith the hydrophobic interactions that hold the t;o αβ dimers together and ;e observe a '6? kDa protein/ Finally5 ;hen ;e add 'mercaptoethanol during D-+AE the disul#ide bonds are broken and ;e observe ' polypeptides o# .63 and >3 kDa/
(evel o# Di##iculty2 oderate ection2 3/'D (earning ob4ective2 Protein Purification and Analysis