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BIBC 110 dutnall midterm 2

BIBC 110 dutnall midterm 2 - Haste “50 sronanr m Question...

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Unformatted text preview: Haste: “50 sronanr m: Question 1. [2i] pts total} The table below shows the amino acid composition, size and behavior on a size exclusion column of eight different proteins. Use the intoonanon to answer the questions below. Elution volume from size L ' t H ? exclusion charge a" F Mt- #hfflfl'fln‘fiE Protein {De} _—_—--I_EEEI I§-_—“£---—-[E- E‘_—--IE_E-IEI_- _—E__ElI-_--EE_EE- 'i _wflfl-_Ell¥fillfl-—EEE- - “l “MEIE- WEE—mflfi_ [a} Which proteins would you expect to bind to an anion exchange column at neutral and in what order would you predict them to elute with an increasing gradient of Natl] conoeotration‘H pts] AeFeBe-l-léél- {in} Which proteins would you expect to bind to a cation exchange column at neutral H, and in what order would you predict them to elute with an increasing gradient of NaCl oottoeno'ation'? 4 pts] EéDLC {c} If you bound the proteins to a hydrottyapatite column, which proteins wouid you expect to elute with an increasing concentration gradient of sodium phosphate and in what order?‘ {3 pts) fit at l: e: E. e: H £1 G- (d) Describe two reasons that can explain the behavior of protein H during size exclusion chromatography. [4 FEE] Prgkflil"! H lift filmslfl.’ IF” Prfilfllfi G Vt. (and Holt—tel. ill '45.) Flt-titan Vtfl, Elf. Mr 50 Prfidifllfi Hr [Dialect-n H fusions-l M Pink-lat:- Ell.— fir Gl'fiL-fi. G '. 2 K Hf- FULL-1A H . Be mtg” H m‘tafil he ea dimer (3,) or non- spine rte otl Wt” (I) A researcher (wants to purify protein F but finds that fractions from an ion exchange column that contain protein F always contain a significant amount of protein B. So they apply fliese fractions to a hydroxyapatite column but again find that fractions containing protein F are still contaminated with protein B. They then realise that there is simple way to obtain pure protein F from the hydroxyspatite fractions when they learn that protein B is a l-tinase enzyme tltat binds ATP. is} Why did the ion exchange and hydroayapatite columns fail to separate proteins F and B? {2 pts} ‘1‘} Choral. Proteus F Simllar ‘l'u pied-em E3 (—0: us. -Il) if) What chromatography method did they select after these columns failed and how did they use it to purify protein F? {3 pts} fiFfiolla ChroMotltxjrufebtj (l) Protein E3 Loki: Pia—1F,So CDUoJ-uenl’tj allaelx PEEP to Elel'imorfl Pheasa— mll MG" RULE-5 iju-Et" F4133, 3 mill- latexfilr F mall He'll“ 5::- will POLE!- {bfll’l ColLthl' mlxocl' pans-ca Harts —e pore Ft ("0 NeiME: STUDENT ID: Question 2 (It! pia total]. {a} Briefly describe the physical basis for analysis of molecular interactions via chemical cross-linking and SD5-pciyacrylaniiee gel electrophoresis. {4 pts) SD51 denature-s Mp'lEL—u'fifl 5 - di 5 rv'f'f‘l'r amt-ore: out] inherucciien‘s bet-Ema _ . mate: i. 1.1-1! 50 indie idea-J Prat-ems Ci, complain. on.“ EGfEJi-L“: ‘ IL . ‘ . +run MLmdirfl lit-Hr Cmfie-Jlm flhflflfb Jenna Proteins .Lcibg treachxm‘ in. m compute SI)? lit-$1“ coir latte-I“: LbJolm‘i- loam} 5c- migrate. ct Ht“ 5cm 0+ Lcmpfm’l‘s C “:55 - Itfih-ed (h) The interaction between three proteins (A. B and C} is analyzed using chemical cross-linking and SD5- PAGE. The results of the analysis are shown schematically in the picture below. The size (M, in hills) of each hand visible in the gel is indicated above the hand. Three different mixtures were analyzed as follows: {i} Protein A alone is analyzed without cross-linking (No X-Iink) either Twithout {-BMEJ or with [+BME) a reducing agent, and then after complete cross-linking in the presence of reducing agent {with X-link, +BlvaLJ. (ii) A mistere of protein 1.?er n 2.5 kfinl and protein C (M, = it] His) is analyzed in a similar way. (iii) A minors of protein A. B and C is analyzed. In this case the samples were analyzed only in the sence of reducing agent, either without cross-linking or with cross-linking for two different petiods of time I: or 120 minutes]. It is detennined that after so min. each molecule of complex contains at least 4 cross-links and that there is complete cross-linking after 111] min. Use the results of the analysis to deduce the composition and relative spatial orangement of the proteins for each mixture (1. ii, iii}. Use pictures to illustrate your answers. Indicate the presence of any special find of molecular interaction that may be present. " f3?”- iflkD-e .? 'lc'lfi'iy‘f‘ide tan-mam mammmcinnwumsawc [flPl'DtfiiflH-GPISJ: 4.3.“; tut-L0H- - Ian I I ”aim“ :m Hex-en m "a #11:” +Khflt+gflé 10km... din-MK (11'2“?) we outshone one one was am mm min _5 @356.) .. [ii] Protein B + C (4 pts}: Ne all“ +/— Bus .'.r1o difieifide W £75- ‘tc+2.5’ so helereciimer Ll. ' . '. W JUL .1 "was: {iii} Protein a + a + c {s pts): lZDmirt : can is lot refs (-9 Ronnie lSolse 23(g+;)+ l. (ZR):Z:LS + 1c- ‘2 E-irC No ice; no So innit (3+E) or smoke) W5 1 EH; hEWGd‘tmtf’: mit- £155.: hafifld’f' - Nchr; no UnofUS'E—finhzd Ft era @‘3 meantime” Ste. C Cid-lint. 5:: Ital- cieoe'fi.» P1 is @0‘®®© {c} What is the composition of the T5 kDa, 35 ltDa and lltl ltDa bands observed after 5!] min. cross-linking of the complex {3 pts}? - Time: the)“: BSkDa: mam“: llama: 969 {gt NM 9993 (atlas) G‘Qo‘afl title. t .‘L-fii . NM: STUDENT lIJI: Question 3. [EDI pts total} {a} Draw a schematic diagram of a time of flight {TOP} mass spectrum analyzer. Efleflx describe the physical basis by which molecular ions are analyzed. [I5 pta] L..—..—| Dr; ‘l— P tfln 1-. Emit-L] No gr: 0:135“ {on kineht enofitj iii-rm! £53m" “”1 TIFFL hit-ken £3- ‘Li’atdet'ill tif'l{-+ ESE-“N :tiie-l'euttcf'f in am Hie-eh“ ,3; Wm M tins: "in-is enters dril‘l- my fighter! {b} it research student uses mass spectrometry to determine the mass of a purified protein. The measured mess of the tein is 11,635.15 Do but based on amino acid sequence the protein is predicted to have a mass of 1 1,233??? He. The student decides that the protein must have been covalently modified. Using the data below show how titre}.f determined the nature and extent of the modification. (5 pts) Host. cltl'kreoot = ileeS-e-ttennrs = 49¢;qu Modification Isotopic rnsss . . changg (D3). d'fFfl-afifl J5: Lhfll't? far tmgdttl-tt-nihm Aeetplation 42.01 Hood {qt 2- ct * ‘i . ‘5 K ADP-obosylation 541.06 too lat-:3 Memplation 15.04 true—Ijt‘i'E-‘i = 25-6- It Phosphorylation 79.91 ow. free.“ e 5- oo -d=_-' in team it. Nature of modification: Phoephogloj on Number of modification sites: 5 {c} What other possible conclusion[s} can be drawn if a difference between the predicted and measured mass of a protein is observed? (3 pts] Muiuhetrt ct Prob-2t}. SECIJEI‘LCP. a, Prairfolflsitfo (d) Briefly.' describe an eaperimenta] a soft that could be used to test whether the protein had been modified as the student concluded in part {b} er than the mass difference being due to other reasons. Describe the result you would expect to see if the student was right. {15 pts) lfioeicc'h'tc emote m Sept-mini! Ila—j FIIW 5} fataliEin m rqagfllqlafi Phoefiho legal thtr‘l. Le't'tl MVL lowdr g; (more. ontdiQSW-lu Domes-it Lfllfil gruff-n . Nanci unmodified {retain ooo Camper-ism CDJJK U'StJE Phfiiioh-Al‘hee ED rust-wt Phoephedtes ‘c—r—sfiz—f—ij'takfi ME” '0ka imfljhufite boll-erm'lr‘iitt {112(3le .._._—— H a... ti.- them . mi o no. 9. We Wei“ 3 3“?“ 36.10%) fro?“ unmod'. 'J ptflefi. NM: STUDENT ID: Question 4. {2t} pta total} (a) Which of the following molecules absorb light in d1e UV - visible wavelength range of the eiectromagnetic spectmm'? Rank them in order of increasing wavelengd'i of their absorbance maximum, km? {3 pts) {iJCH1=CH-CH=CH1 {ii} CH:=CH-CI-I;H3H=CI-I; {iii} CH;=CH—CH=CH-CH=CH2 (is) CH3-CI-Ig-CH1-CH3 2 con‘ and 3 2 dam midi. n J 3. cmd“fi”*" JD-Jbl-t. m5 ne cinema onsets diam ‘3 one” {fink :L 1:. Liti' rat-ta: MOD 4 (-‘J 4 (it? {b} Which anuno acids contribute most signi icantljyr to absorption of light by a protein if it = 250 anti' [2 pts) { it [lit WP \/ )(less set-5 to] Sketch the Morse [infirm showing the electronic and vibrational energyr levels of the excited state and ground state of a typical uoro hore moiecule. Indicate the transitions that occur during absorption and emission of a photon of light. hE’hat is the relationship between the energy.f and wavelength ofthe absorbed and emitted photon'iI (5 pts} d in’t nut. (d) Describe with the aid of pictures how an ertrinsic lluorophore such as l-anilino-E-napthalene sulfonate {ANS} can be used to detect interactions between molecules [4 pts} I ____.__.—h3 ' If a ti tn 6"th Surface 6 E @ESS acre-aerial; Etienne! fat-dens ' [1155an {his bfiitrg Dims trials in 'mletu Les s’no Fltioresrontt iniflencktih sic-r etc-L _-; proteins Flu-steers [e] Draw the absorption and emission spectra for a donor fluorophore and an acceptor fluorophore such that the process of fluorescence resonance energyr transfer (FRET) could occur between them. {5 pts) {f} 1What other condition must be satisfied for FRET to occur between a donor and acceptor fluorophore'? [1 pt} C1058 i’oxeikar in Space (Mia?- 4 lOD H NAME: STUDENT lIi: Question 5. {2|} pta total) The following diagram shows the forces acting on a particle in the sample cell of a centrifuge. a Cl) {a} Name the three forces acting on the particle (3 pts} ”-21- - -. _ . ' ' * Meniscus STEiZES Lth' (IE éxnfl) (c) Indicate on the sketch below the name and direction of the two processes that are equally balanced at equilibrium in a sedimentation equilibrium experiment. Also indicate the distribution of a macromolecule in the centrifugation chamber at equilibrium. (5 pts] Axis of rotation :3 SE‘hmefisfi—nw afi-*—————'—‘ 'Dt F Winona (d) Match each of the following methodi'phenomenafpropeny on the left to the most appropriate statement in the list to the right. Indicate the statement number in the space provided. (1 pt each, It] pts total] Statement number Statement Ion exchange chromatography 1+ Quantum yield MAIDI 2. A = eel Mass sneetrotnetryr 3. MAI-EpL-‘RT Electrophoresis 4. U‘Wyiaible absorption Lambert-Beer law 5. Separation based on charge Isoelectrie focusing 5. Fluorescence Relaxation by non-radiative transition 7. Separation based on chargefmass Relaxation by radiative transition 3. Molecular ionization method Quenching 9. Separation based on size lwlrli‘hyhhbmlm Sedimentation coefficient 1t]. Separation based on massfcharge ...
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