Otázky k predmetu Proteíny, štruktúra a funkcia Katedra...

Otázky k predmetu Proteíny, štruktúra a funkcia Katedra biochémie, ÚCHV PF UPJŠ, 2016

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1. How many ionized forms of lysine can exist in a solution? Draw a structure of a dominant form at pH 5, if pKLys(α-

COOH) = 2.2, pKLys(α-NH3+) = 8.9, pKLys(ε-NH3

+) = 10.3? (2p)

2. The mixture of amino acids consisting of Arg, Cys, Glu and Leu is applied to the strip of paper and subjected to

electrophoresis at pH 7.5. In which direction will these amino acids move (to negative or to positive electrode), if you

know that:

Arg: pK(α-COOH) = 2.2, pK(α-NH3+) = 9.0, pK(Gua+) = 12.5,

Cys: pK(α-COOH) = 1.7, pK(α-NH3+) = 10.8, pK(–SH) = 8.3,

Glu: pK(α-COOH) = 2.2, pK(α-NH3+) = 9.7, pK(γ-COOH) = 4.3,

Leu: pK(α-COOH) = 2.4, pK(α-NH3+) = 9.6? (2p)

3. The solution of a polypeptide was divided into two fractions – the first fraction was subjected to degradation by cy-

anogen bromide (CNBr) while the second fraction to degradation by trypsin. The resulting fragments are as follows:

I. after cyanogen bromide degradation: a) Asp-Ile-Lys-Gln-Met

b) Lys

c) Lys-Phe-Ala-Met

d) Tyr-Arg-Gly-Met

II. after trypsin degradation: a) Gln-Met-Lys

b) Gly-Met-Asp-Ile-Lys

c) Phe-Ala-Met-Lys

d) Tyr-Arg

What was the primary structure of the entire polypeptide chain before degradation? (1p)

4. Select the one amino acid out of each of the following four-amino acid groups that does not belong there and explain

why it is so: (1p)

a) Val, Ala, Leu, Asn e) Cys, Ser, His, Gly

b) Gly, Asp, Glu, Phe f) Met, Glu, Asn, Ile

c) Lys, Arg, His, Leu g) Val, Lys, Cys, Gly

d) Trp, Phe, Tyr, Met h) Ser, Cys, Gln, Glu

5. Calculate percentage of different ionized forms of glycine at pH 3 and 11, if you know that pKGly(α-COOH) = 2.3

a pKGly(α-NH3+) = 9.6. (2p)

Hint:

[pH 3: 16.6% fI, 83.4% fII, 0% fIII; pH 11: 0% fI, 3.8% fII, 96.2% fIII]

6. There are two ways of peptide bond formation between Gly and Thr. Draw structures of both variants. (2p)

Identify the dipeptide with the value of the isoelectric point equal to 6.38, if you know that: (1p)

Gly: pK(α-COOH) = 2.34 and pK(α-NH3+) = 9.60,

Thr: pK(α-COOH) = 2.63 and pK(α-NH3+) = 10.43.

7. Estimate the net charge of the peptide Arg-His-Gly-Phe-Gly-Glu-Lys-Tyr-Cys-Ala at pH 5 and 9, if pKArg(α-

NH3+) = 9.0, pKArg(Gua+) = 12.5, pKHis(Im+) = 6.0, pKGlu(γ-COOH) = 4.3, pKLys(ε-NH3

+) = 10.3, pKTyr(–OH) = 10.1,

pKCys(–SH) = 8.3, pKAla(α-COOH) = 2.3. (2p)

8. The mixture of amino acids Gly, Ala, Glu, Lys, Arg, Ser has been deployed to the cation exchange column in the

presence of 0.1 M HCl. Which amino acids are likely to bound to, and which will elute from the column? The column

was then washed with buffer with pH 6. Which amino acids will release and which will remain bound to the column?

(4p)

9. The mixture of amino acids Cys, Glu, Ser, Ala, Lys, His has been deployed to the anion exchange column at pH 11.

Which amino acids will remain bound to the column? (2p)

10. The mixture of amino acids has been deployed to the cation exchange column at pH 1.0. Elution was performed

with a gradient of increasing pH. Estimate the order of elution of the amino acids: Gly, Asp, Tyr, Ala, His, Arg.

(1p)


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11. The following compounds are often used in the study of proteins:

a) CNBr b) urea c) mercaptoethanol

d) 6 M HCl e) ninhydrin f) 2,4-dinitrofluorbenzene

A) Which of them is used to label NH2 terminus of the protein? (1p)

B) Which of them is used to cleave the peptide bond at the carboxyl-terminal Met? (1p)

C) Which one is used to cleave the intermolecular and intramolecular disulfide bonds? (1p)

12. In the reaction of Lys with 2,4,6-trinitrobenzene sulfonate a product with absorption maximum at 367 nm is gener-

ated. Is it possible to apply it somehow? (1p)

13. Which of the 20 encoded amino acids is the smallest in the volume and which one is the largest? (1p)

14. Draw the reaction scheme demonstrating the change of a charge at the side chain of Lys from positive to negative.

(2p)

15. Draw at least one reaction scheme for the reduction of a disulfide bond. (2p)

16. Name three techniques which make it possible to determine the size of proteins. (1p)

17. Which of the encoded amino acids contains indole group in its structure? (1p)

18. Which of the encoded amino acids is a mixture of protonated and deprotonated forms at physiological pH? (1p)

19. What is the difference between gel filtration and gel electrophoresis? (1p)

20. Which salt is commonly used for the precipitation of proteins? (1p)

21. Gramicidin is a heterogeneous mixture of several antibiotic compounds with the following common structure:

formyl–L-X–Gly–L-Ala–D-Leu–L-Ala–D-Val–L-Val–D-Val–L-Trp–D-Leu–L-Y–D-Leu–L-Trp–D-Leu–L-Trp–

ethanolamine

Provided that X can be Val or Ile, and Y can be Trp, Phe and Tyr,

a) indicate how many different pentadecapeptides are present in the mixture, (1p)

b) give the exact molecular weight of the peptide containing Ile and Tyr in place of X and Y, respectively, (1p)

c) determine the net charge of the peptide at pH 7, if X is Val and Y is Tyr. (1p)

[a) 6 b) 1 871 g/mol c) 0]

22. How can you use performic acid in relation to proteins? (1p)

23. What is the yield (in %) in the synthesis of a decapeptide, if the efficiency of the right amino acid connection during

each step is 80 %? (1p)

0.810 = 11% [Proteins, p.45] alebo 0.89 = 13% [Lehninger, p.106]

24. Briefly describe each step in the synthesis of a peptide on a solid surface (reaction schemes are not necessary).

(2p)

25. What is the gel filtration for in relation to proteins? (2p)

26. Reaction of hydrazine with Arg (hydrazinolysis) results in a cleavage of the basic group of Arg and production of

Orn. Draw the reaction scheme. (2p)

27. Name the four problems/disadvantages that accompany peptide synthesis. (2p)

28. What is the major difference between synthesis of peptides and protein biosynthesis? (1p)

29. What is the purpose of activation of the amino acid carboxyl groups during synthesis of peptides? (1p)

30. The reaction of performic acid with carboxymethylCys results in sulfone production. Draw the reaction scheme.

What can be the reaction used for? (2p)

31. What can be the reaction of Cys with iodine acetate and iodoacetamide used for? (1p)

32. Cysteic acid is produced by reduction of disulfide bond by performic acid. Draw the reaction scheme. What can be

this reaction used for? (2p)


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33. A protein was unfolded by a strong inorganic acid. How many % of its unfolded form is present in a solution at

pH 4.0, if the value of apparent pK is 3.52? (1p)

a) 0% b) 25% c) 50% d) 75% e) 100%

34. What parameter is crucial for separation of ions in mass spectrometry? (1p)

35. Name the essential parts of the mass spectrometer and indicate their functions. (2p)

36. What is the essential criterion for ordering the ions in the Hofmeister series? (1p)

37. What is the purpose of chemical modifications of amino acids in vitro? List at least three reasons. (1p)

38. Give examples of chemical modifications of any two amino acids along with their applications. (2p)

39. What information gives us the Ramachandran diagram? (1p)

40. Formation of a polypeptide chain during protein biosynthesis is a complex process that can be divided into several

steps. List them and briefly describe. (2p)

41. What is the principle of isoelectric focusing

capillary electrophoresis

SDS PAGE

TLC? (1p)

42. Draw a formula of the following tripeptide: Phe-Met-Arg

Pro-Ala-Asn

Ser-Val-Glu

Trp-Ile-Thr. (3p)

43. Draw a formula of the following dipeptides so that the cysteine residues were linked by a disulfide bond:

Val-Cys a Cys-Pro (4p)

44. Which of the encoded amino acids contain the benzene ring? (3p)

45. Give the two examples of the fibrous proteins. (2p)

46. Arginine contains strongly basic group whose basicity is comparable to that of NaOH. What is its name? (1p)

47. What is the net charge of Lys at pH 7, if pKLys(α-COOH) = 2.2, pKLys(α-NH3+) = 8.9 and pKLys(ε-NH3

+) = 10.3 ?

(1p)

48. Which enzyme or chemical substance can be used to cleave peptide bond adjacent to the following amino acid resi-

dues: Arg

Lys

Met

Phe? (1p)

49. Cyanogen bromide (CNBr) hydrolyzes peptide bonds at the C-terminus of ….. (write appropriate amino acid) (1p)

50. Trypsin is a highly specific enzyme that catalyzes the cleavage of peptide bonds on the carboxyl side of two amino

acid residues. Write their names. (2p)

51. There is a polypeptide with the following sequence: Cys-His-Arg-Leu-Gly-Ala-Val-Phe-Ile-Glu-Asn-Tyr-Asp-

Pro-Met-Trp-Ser-Lys-Gln-Asp.

How many different fragments can be obtained after complete digestion by pepsin

trypsin

chymotrypsin

CNBr?

Explain your answer. (2p)

52. Estimate the number of amino acid residues in a protein with MW of 15 000 g/mol? Assume that it does not contain

any prosthetic group. (1p)

[15 000/111 = 135 AK]


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53. What is the product of the Edman’s degradation of proteins? How can be identified? (1p)

54. Which of the three assay tubes containing protein albumin (tube 1), the polypeptide insulin (tube 2) and the amino

acid Ala (tube 3) will be positive for biuret test? Explain your answer. (2p)

55. A solution contains three different globular proteins: cytochrome c (MW = 12 400 g/mol), ribonuclease A

(MW = 13 700 g/mol) and myoglobin (MW = 17 000 g/mol). Arrange them according to the order of their elution

out of the chromatographic column in gel filtration. (1p)

56. In addition to L-amino acids that generally form peptides and proteins, D-amino acids can also be found in organ-

isms. Give an example where can be found? (1p)

57. What is a role of D-amino acids in organisms? (1p)

58. Draw a structure of tripeptide glutathione, if you know its primary structure: γ-Glu-Cys-Gly. (3p)

59. A sample contains three different globular proteins: cytochrome c (MW = 12 400 g/mol), ovalbumin

(MW = 44 000 g/mol) and myoglobin (MW = 17 000 g/mol). Arrange them according to their electrophoretic mo-

bilities after separation by SDS PAGE. Start with the protein that is closest to a negative electrode. (1p)

60. What is a product of the spontaneous cyclization of Gln at the amino-terminus of a polypeptide? (1p)

61. List the wavelengths of the absorbance maxima of aromatic amino acids? (1p)

62. What is the wavelength range of peptide bond absorption? (1p)

[200-215 nm, 190-230 nm]

63. Write the name of the following peptide: (2p)

OH

CH2

NH2

CH2

CO NH CH CO NH CH CO NH

CH2

CH

CH2

COOH

CH2

S CH3

CNH

N

64. What are the functions of glutathione (GSH) in organisms? (2p)

65. Write the units of the molar absorption coefficient (ε)? (1p)

66. Explain the term emission spectrum of a protein. (1p)

67. What is the principle of fluorimetry? (1p)

68. Explain the term fluorescence quenching. (1p)

69. Provide at least one way of how to quench the fluorophore (for example Trp) fluorescence emission. (1p)

70. Draw a reaction scheme of amino acid activation and its connection to the tRNA in the protein biosynthesis. (3p)

71. How many classes of aminoacyl-tRNA synthetases are known and what is the criterion for their division? (2p)

72. A solution contains a peptide with concentration of 2 g dm-3. Its transmittance in 1-cm cuvette is 75% at 457 nm.

a) Calculate transmittance of the solutions with the following concentrations of the peptide: 4 g dm-3, 1 g dm-3

a 6 g dm-3.

b) Molecular weight of the peptide is 250 g/mol. Calculate its molar absorption coefficient at 457 nm. (4p)

[a) 56.2 %, 86.6 %, 42.2 %; b) 15.6 mol-1 dm3 cm-1 = 15.6 M-1 cm-1]

73. Transmittance of a protein solution with a concentration of 1x10-5 mol dm-3 is 0.702 at 260 nm in 1-cm cuvette.

Calculate:

a) transmittance of the solution in 3-cm cuvette,

b) absorbance of the solution in 3-cm cuvette,


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c) absorbance and transmittance of a protein solution with the concentration of 5x10-5 mol dm-3 in 1-cm cuvette.

(4p)

[a) 34.6 %; b) 0.461; c) 0.768, 17 %]

74. Calculate absorbance and transmittance of the following protein solutions at 260 and 340 nm in 1-cm cuvette:

a) 2.2x10-5 M of protein A,

b) 7x10-6 M of protein A and 4.2x10-5 M of protein B.

Molar absorption coefficients are as follows: 15x103 M-1 cm-1 at 260 nm and 6220 M-1 cm-1 at 340 nm for protein A,

and 15.4x103 M-1 cm-1 at 260 nm and zero at 340 nm for protein B. (4p)

[a) at 260 nm: 0.33, 46.8 %; at 340 nm: 0.137, 73 %; b) at 260 nm: 0.752, 17.7 %; at 340 nm: 0.044, 90.5 %]

75. A protein solution has the specific absorption coefficient (1%

1cmA ) of 0.2 at 450 nm. Calculate the protein concentra-

tion in the solution, if its absorbance is 0.38 in 3-cm cuvette. (2p)

[c = 0.633 %]

76. A solution of human serum albumin with a concentration of 1 mg/mL and an absorbance of 0.56 at 280 nm (1-cm

cuvette) was used as a standard. A sample of unknown proteins has an absorbance of 0.12 at 280 nm (1-cm cuvette).

What is the concentration of unknown proteins in the solution? (2p)

[c = 0.21 mg/mL]

77. An unknown protein solution (0.3 mL) was diluted by a water (0.9 mL). A volume of 0.5 mL of that diluted solution

was mixed with 4.5 mL of a biuret reagent. An absorbance of this solution was 0.18 at 540 nm (1-cm cuvette). The

same volume, i.e. 0.5 mL, of a standard protein solution (4 mg/mL) had an absorbance of 0.12 following the addi-

tion of 4.5 mL of the biuret reagent. Calculate the concentration of unknown protein before its dilution. (2p)

[c = 24 mg/mL]

78. A molybdenum containing ferredoxin is a tetrameric enzyme involved in nitrogen fixation process. Its 0.1% solution

has an absorbance of 1.5 at 280 nm (1-cm cuvette). A sample of this enzyme was prepared with the molybdenum

content of 20.1x10-6 g/mL; its absorbance was 0.375 at 280 nm after 50-fold dilution. Calculate the minimum mo-

lecular weight of that enzyme. The relative atomic mass of molybdenum is 95.94. (2p)

[c(Mo) = 2.095x10-4 M, c(protein) = 12.5 mg/mL, MW(subunit) = 59 700 Da, MW(tetramer) = 239 000 Da]

79. What is the principle of affinity chromatography? (1p)

80. What information about a protein can be obtained by differentiating its absorption spectrum? (1p)

81. How many extremes will have a symmetrical single-peak spectrum of a protein after using the first derivative?

Draw the original spectrum and its first derivative. (2p)

82. A protein containing amino acid Trp was excited with a 280-nm beam. Which of the following wavelengths is the

one you would expect the maximum in the emission spectrum of the protein at? Explain your choice.

a) 210 nm b) 280 nm c) 350 nm (2p)

83. Which of the amino acids is essential for formation of selenocysteine (Sec) in organisms? (1p)

84. Name two enzymes involved in the synthesis of glutathione. (2p)

85. Explain the term torsion angles in proteins. How are torsion angles marked? (2p)

86. Give the ways of how proteins and peptides are synthetized in organisms? (2p)

87. Where are the proteins destined to be exported from the cell synthetized? (1p)

88. What type of a post-translational modification is catalyzed by:

deformylase

Met-aminopeptidase

arginyl tRNA-protein transferase

Nα-acetyltransferase

prolyl 4-hydroxylase

lyzyl 5-hydroxylase

kinase

phosphatase


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ADP-ribosyl transferase

protein-disulfide isomerase? (1p)

89. Which amino acids may be post-translationally phosphorylated? (3p)

90. Which amino acids may be post-translationally hydroxylated? (2p)

91. For which protein is the hydroxylation of Pro and Lys typical? (1p)

92. List the places (organelles, etc.) within the living cells where enzymatic post-translational modifications occur?

(1p)

93. In relation to proteins, what is the significance of

N-terminus myristoylation

N-terminus acetylation

C-terminus amidation

glycosylation

phosphorylation

ADP-ribosylation? (1p)

94. Draw the structure of glucosyl serine. (1p)

95. List the types of enzymatic glycosylation? (2p)

96. Which enzyme catalyzes tyrosine sulfation in the Golgi apparatus? (1p)

97. In which place of a cell following processes occur:

disulfide bond formation

tyrosine sulfation

alpha amino group myristoylation? (1p)

98. Define the term zymogen? (1p)

99. What feature is necessary to specify the amino acid for enzymatic post-translational modification? (2p)

100. Draw one cycle of a reaction between tripeptide Ala-Gly-Cys and Edman’s reagent. (2p)

101. Which information about proteins can be obtained by the diagonal (two-dimensional) electrophoresis? (1p)

102. Which of the following statements obout proteins are wrong? (1p)

a) Information about the primary structure of proteins is stored in the primary structure of nucleic acids.

b) The primary structure of proteins determines their tertiary structure.

c) Only the proteins are able to catalyze reactions in organisms.

d) Proteins destined for export from the cell are synthesized on the endoplasmic reticulum.

e) Glycoproteins are biomolecules with covalent linkage between proteins and carbohydrates.

103. Name the reaction that is widely used for detection of amino acids and peptides on TLC plates? (1p)

104. Which enzyme catalyzes activation of an amino acid and its attachment to tRNA? (1p)

105. Which enzyme catalyzes formation of a peptide bond in protein synthesis? (1p)

106. What are the functions of initiation factors IF1, IF2 and IF3 during protein synthesis in prokaryotes? (3p)

107. Write the relationship between a molecular weight of a protein and its electrophoretic mobility in SDS PAGE.

(1p)

108. There is a following relationship between an elution volume (Ve) and a molecular weight (MW) experimentally

found by a gel filtration for series of globular proteins: Ve = 547 – 99.5 logMw. What is a molecular weight of an

unknown protein if 100 mL of a buffer was needed to elute it from a column? (1p)

109. Explain the role of SDS in protein separation by PAGE. (1p)


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110. Name the parts of a signal peptide of a protein and explain their functions? (2p)

111. Briefly outline the steps of a protein biosynthesis on the endoplasmic reticulum. (3p)

112. List four functions of chaperones in organisms. (2p)

113. What classes of molecular chaperones are known? (1p)

114. Which enzyme catalyzes cleavage of a signal peptide from a polypeptide chain in the endoplasmic reticulum?

(1p)

115. List four enzymatic post-translational modifications that occur in the endoplasmic reticulum. (2p)

116. Provide two functional groups of amino acids that are most attacked during non-enzymatic post-translational modi-

fications. (1p)

117. What is the molar absorption coefficient of a protein at 422 nm, if you know the following data:

Abs. at 422 nm 0,154 0,493 0,832 1,186

[Protein] (μmol dm-3) 1 3,2 5,4 7,7

A 1-cm quartz cuvette was used. (1p)

118. What information about a protein can be obtained by absorption spectroscopy

circular dichroism spectroscopy

differential scanning calorimetry

gel filtration? (1p)

119. Which of the amino acids are essential for determination of proteins concentration using absorbance at 280 nm?

(1p)

120. Twenty-second proteinogenic amino acid (pyrrolysine, Pyl) was discovered in: (1p)

a) Escherichia coli b) Methanosarcina barkeri c) Streptococcus thermophilus

121. Explain the principle of determining the molecular weight of proteins by

SDS PAGE

gel filtration

analytical ultracentrifugation

mass spectrometry. (1p)

122. List amino acids that form the following peptide: PRQTEINY (1p)

123. How many arginines are in the following primary structure (PDB/1LYZ)? (1p) KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDY

GILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL

124. What can you write about the protein with the following absorption spectrum? (1p)


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125. What information about protein can we get from its differential absorption spectrum? (1p)

126. The following absorption spectra belong to the same protein. So, what makes the difference? (1p)

127. Which of the cell organelles contains cytochrom c oxidase? (1p)

128. List the functions of cytochrome c in organisms? (1p)

129. Which techniques are suitable for 3D structure determination of proteins? (1p)

130. Calculate the length of α-helix (in nm) formed by a polypeptide chain of twenty amino acids, if you know that

there are 3.6 amino acid residues per turn and the height of one complete turn is 5.4 Å? (1p)

[30 Å = 3 nm]

131. Define the following terms quaternary

tertiary

secondary

primary structure of proteins? (1p)

132. List non-covalent interactions that stabilize tertiary structure of proteins? (2p)

133. What impact on the pK value of the Glu side chain will have the presence of the positive charge of the Lys side

chain? (1p)

134. Which thermodynamic parameter is a measure of the protein stability? (1p)

135. Which of the following requires less energy: to break down the covalent C–H bond in a methane molecule or to

denature a protein? Explain your answer. (1p)

136. Which type of non-covalent interactions is predominant in the stabilization of α-helices in proteins? (1p)

137. Define the term amphipathic α-helix. (1p)

138. Which of the amino acids increases the rigidity of a polypeptide chain the most and why? (2p)

139. Explain how ionic interactions

van der Waals interactions

hydrogen bonds

hydrophobic interactions are formed in proteins. (1p)

140. Draw a plot of potential energy versus internuclear distance. (1p)

141. Explain why extremely high or low pH values destroy the native protein structure. (1p)

142. There are methyl group of Ala and carbonyl group of a peptide bond close each other in a protein. What type of

non-covalent interaction between the two can be expected? (1p)

143. The side chains of which amino acids will mutually form ionic interaction? (1p)

144. Define the term protein denaturation. (1p)


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145. Explain the mechanism of protein denaturation by denaturing agents. (1p)

146. List the four basic thermodynamic models of the transition of simple proteins from native to denatured state. (2p)

147. How can be proved that a protein conformational change is a simple two-state transition? (1p)

148. Why the heat capacity of the thermally-unfolded protein increases as compared to its native form? (1p)

149. List the parameters that uniquely characterize the so-called S-curve of the thermally-induced unfolding of a pro-

tein. (2p)

150. What are the main characteristics of a/an α-helix

β-pleated sheet

π-helix

310-helix

β-turn

Ω-loop? (1p)

151. List the types of supersecondary structures? (2p)

152. What structural characteristics is typical for α-proteins

β-proteins

α/β-proteins

α + β-proteins? (1p)

153. What types of intramolecular movements can be distinguished in proteins? (2p)

154. List the basic models of protein folding along with their characteristics. (2p)

155. List the main characteristics of the so-called unstructured proteins. (2p)

156. What are amyloids (amyloid fibrils)? How are they formed? (2p)

157. What are prions and what forms are distinguished? (2p)

158. What makes it possible to diagnose pathogenic form of prion? (1p)

159. Why are amyloid structures dangerous for human beings? (1p)

160. Urea-induced denaturation of protein at constant temperature was studied. The value of D1/2 was found to be 6 M.

Calculate the value of Gibbs free energy change in the absence of urea, if the value of m is equal to 5 kJ mol-1 M-1.

(1p)

161. By urea-induced unfolding of protein A the values of D1/2 and m were found to be 5 M and 5 kJ mol-1 M-1, respec-

tively, at 25 °C. Similarly, for protein B the values of D1/2 and m were found to be 6 M and 5 kJ mol-1 M-1, respec-

tively. Which of the two proteins is more stable at given temperature? (1p)

162. Which of the following combinations of the torsion angles Φ and Ψ is typical for the right-handed α-helix? (1p)

a) -57, -47 b) -57, +47 c) +57, -47 d) +57, +47

163. How are the transmembrane proteins incorporated into the cell and organelle membranes? (1p)

164. List the ways of attachment of peripheral membrane proteins to the cell and organelle membranes? (2p)

165. How can be the rate of protein degradation determined in vivo? (1p)

166. Define the term ubiquitins. What is their function? (1p)

167. Which of the amino acids is essential for binding of a protein to be degraded to ubiquitin? (1p)

168. List the essential amino acids for humans. (1p)

[Thr, His, Lys, Ile, Leu, Met, Phe, Trp, Val]


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169. What could cause protein degradation? (1p)

170. Transition temperature of a protein was found to be 80 °C by DSC. Transition temperature of the same protein

determined by absorption spectroscopy at 390 nm was found to be 60 °C. Based on this information, what can you

conclude as to the mechanism of the protein unfolding? (1p)

171. Explain the term protein folding. Which interactions are essential during this process? (2p)

172. Which of the amino acids can be generally found inside of the globular proteins? What do these amino acids have

in common? (2p)

173. Which of the amino acids can be generally found outside of the globular proteins? What do these amino acids have

in common? (2p)

174. Which of the following generally leads to stabilization of a protein structure? (1p)

a) disulfide bond formation,

b) replacement of two amino acids forming a hydrogen bond by two glycines,

c) replacement of a small amino acid in the tightly packed hydrophobic core by tryptophan.

175. A protein that interacts with a negatively charged substrate was isolated. At the same time it was found that the key

part of the protein responsible for this interaction is a part of the following sequence: ALMSQRTWNGKCP.

Which of the amino acids in this sequence would attract your attention if you would like to create a mutation in

this protein? (1p)

176. Which of the following amino acids – Asp, Glu, Lys, Ser, Tyr – would most probably bind pyruvate in the enzyme

pyruvate dehydrogenase? Explain your answer. (2p)

177. Which of the following amino acids – Asp, Glu, His, Lys, Arg – would be the most suitable for the acid-base ca-

talysis at neutral pH? Explain your answer. (2p)

178. What function performs lysozyme in the body? (1p)

179. Give the three amino acids that form chromophore in GFP (green fluorescent protein)? (1p)

Ser65-Tyr66-Gly67

180. Which of the following proteins has no quaternary structure? (1b)

a) cytochrome c b) myoglobin c) hemoglobin d) cytochrome c oxidase

181. Calculate the ratio of denatured (D) to native (N) states of a protein at 25 °C, if you know that ΔGD(25 °C) of that

protein is 28 kJ/mol. Assume, that the denaturation is reversible and there are no intermediates formed. (1p)

[1.2x10-5 (D) k 1 (N)]

182. Which of the following does not belong to the group of membrane proteins? (1p)

a) albumin b) lysozyme c) cytochrome bc1 d) myoglobin

183. Combine the correct pair of amino acids and type of bond/interaction between them that can be expected: (1p)

A. Leu and Val 1. disulfide bond

B. Cys and Cys 2. ionic interaction

C. Asp and Lys 3. hydrogen bonding

D. Ser and Thr 4. hydrophobic interaction

184. What is FRET and how can be this phenomenon used in the study of proteins? (1p)

185. There are two post-translational enzymatic modifications that are related to Cys. Name them. (1p)

186. Explain the term reactive oxygen species (ROS). (1p)

187. Draw formation of ionic bond between the side chains of Lys and Asp

hydrogen bonding between the side chains of Asp and Ser

Van der Waals interaction between the side chains of two Val. (1p)

188. The following values of ΔHvH and ΔHcal for conformational transition of a protein were determined be DSC:

100 kJ/mol and 200 kJ/mol, respectively. Can be this conformational change regarded as a two-state process? Ex-

plain your answer. (1p)


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189. List at least three information about the following protein: collagen

cytochrome c

ubiquitin

cytochrome c oxidase

cytochrome bc1. (1p)

190. Explain the term hydrophobicity scale of amino acids. (1p)

191. Which of the amino acids is essential for the formation of desmosine? (1p)

192. Explain why only quartz cuvettes may be used when measuring absorbance in the UV region? (1p)

193. Three different techniques were used to study a protein. Based on the images below, identify the techniques and

briefly describe what information about the protein can be obtained using corresponding one. (2p)

600500400

1

0.5

0

(nm)

Absorb

ancia

10080604020

10

5

0

T (°C)

Cp (

kcal/°C

/mol)

194. Which of the following triads is a chromophore in GFP? (1p)

a) Ser-Tyr-Gly b) Ser-Trp-Gly c) Ser-Tyr-Glu d) Ser-Gly-Tyr

195. Draw a scheme of Schiff base formation between ε-NH2 of Lys and aldehyde group of glucose. (1p)

196. List at least three proteins which have a structural role in organisms. (1p)

197. Hydrogen peroxide is formed as a byproduct of glucose transformation to glucono-δ-lactone catalyzed by glucose

oxidase (GOX). H2O2 belongs to a group of so-called reactive oxygen species that are dangerous for cells. Explain

why. (1p)

198. How many amino acids comprises human lysozyme

sperm whale myoglobin

cobra phospholipase A2

horse heart cytochrome c

human ubiquitin? (1p)

[1LZ1: 130; 1SWM: 153; 1YXL: 119; 1AKK: 104; 1UBQ: 76]

199. Based on the following CD spectrum, estimate which element of the secondary structure dominates in the protein.

(1p)

A B

C


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200. The following MALDI mass spectrum includes signals of three proteins (cytochrome c, myoglobin, ubiquitin):

Assign each peak to a corresponding protein. Explain your choice. (1p)

(Note: in all three cases, the macromolecular ion charge is +1.)

201. Commercially supplied enzyme glucose oxidase (GOX) contains following information on the packaging:

* isolated from Aspergillus niger * amount: 0.5 MU * α-amylase: <0.05%

* lyophilized * ratio GOX/catalase: >2000 * invertase: <0.5%

* appearance: yellow powder * price: € 153.75 * maltase: <0.05%

* store at -20 °C * activity: 360 U/mg protein (280 U/mg powder)

a) How many % of GOX contains supplied lyophilized powder? (1p)

b) How many grams of lyophilized powder are in one package? (1p)

[78%, 1.8 g]

202. Calculate the value of the molar absorption coefficient (ε) of human lysozyme (PDB/1LZ1) at 280 nm, if you

know the primary structure: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDY-

GIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV

Be sure to include the correct unit. (1p)

[36 440 M-1cm-1]

203. Calculate absorbance of the solution of cobra phospholipase A2 (PDB/1YXL) with concentration of 1 mg/ml at

280 nm (ε2800.1%), if you know the primary structure:

NLYQFKNMIQCTVPSRSWQDFADYGCYCGKGGSGTPVDDLDRCCQVHDNCYNEAENIS-

GCRPYFKTYSYECTQGTLTCKGDNNACAASVCDCDRLAAICFAGAPYNDANYNIDLKARCN

The molecular weight of phospholipase is 13 170 g/mol. (1p)

[1.44]

204. What is the molar concentration of a solution of cytochrome c oxidase (CcO) that was prepared by dissolving of

1 mg of protein in 1 ml solution? The molecular weight of CcO is 200 000 g/mol. (1p)

[5 µM]

205. Provide at least two proteins (protein groups) comprising coiled coil in the structure. (1p)


13

[α-keratín, kinezín, myozín/transkripčné faktory (tzv. Leu-zipsy), membránové proteíny, vláknité proteíny, proteíny

pohybu]

206. The following image shows the absorption spectra of two proteins. What do these proteins have in common and

what do they have different? (1p)

207. Which of the three points (A, B, C) corresponds to albumin, if you know that it consists of 585 amino acids? (1p)

208. What gives yellow appearance to protein glucose oxidase? (1p)

209. Divide encoded amino acids into groups according to the polarity of their side chains. (1p)

210. Provide two techniques, which make it possible to determine the number of subunits in multi-subunit proteins such

as cytochrome c oxidase, cytochrome bc1. (1p)

211. Give at least three non-enzymatic post-translational modifications of proteins which are beneficial to the organ-

isms. (1p)

212. The package of an enzyme contains 10 mg of lyophilized powder with enzymatic activity of 25 U. Calculate spe-

cific activity of the enzyme. (1p)

213. Name at least three examples of cross-links in proteins. (1p)

[Cys-Cys/disulfidová, Lys-Lys/Lys-norLeu+aldolová+(izo)dezmozín, peptidové/Gln-Lys]

214. List amino acids with side chains that are involved in the formation of cross-links in proteins. (1p)

215. The following parameters were determined from thermally-induced reversible unfolding (N D) of a protein:

Tm = 75 °C, ΔHm = 400 kJ/mol and ΔCp = 5 kJ mol-1 K-1. Calculate the value of ΔG for the protein at 25 °C. (1p)


14

Hint:

m

mp

m

mDT

TTTTC

T

THTG ln1)(

[38.6 kJ/mol]

216. Name the cross-link that is specific for a macromolecule of elastin. (1p)

217. Why is a solution of lysozyme colorless? (1p)

218. Given the following peptide: Met-Glu-Arg-Thr-Gly (5p)

a) name N-terminal residue, b) indicate the number of charged groups at pH 7,

c) indicate the total net charge at pH 1, d) calculate the value of pI for the whole pentapeptid,

e) calculate the values of pI for every single amino acid separately.

AK pK(α-COOH) pK(α-NH3) pK(R)

Met 2.15 9.28 –

Glu 2.10 9.47 4.10

Arg 1.83 8.99 12.5

Thr 2.09 9.10 –

Gly 2.35 9.78 –

[a) metionyl b) 4 c) +2 d) 6.69 e) 5.72, 3.10, 10.75, 5.60, 6.07]

219. The protein consisting of 172 amino acids was isolated and purified. There is neither Trp nor Phe in its structure. A

sample of this protein with a concentration of 50 μmol/L has absorbance of 0.447 at 280 nm (1 cm optical path

length).

a) Is there Tyr in its structure? Explain the answer. (1p)

b) If yes, how many? (1p)

c) Calculate the mass concentration of the protein solution. (1p)

The molar absorption coefficient of Tyr is 1 490 dm3 mol-1 cm-1 at 280 nm.

[b) 6 c) 0.96 g/L (mg/mL)]

220. There are structures of four amino acids:

a) Name them. (1p)

b) Indicate those for which the following statements are true: (3p)

i) It has a basic side chain.

ii) It has three ionizable groups.

iii) It is the most hydrophobic of the four.

iv) It has a total net charge of 0 at pH 7.

v) It is formed from essential amino acid Phe in the human body.

vi) It is essential for humans.

[i) D ii) B, D iii) C iv) A, B, C v) B vi) C, D]

Bibliography:

1. Creighton T. E.: Proteins: Structures and Molecular Properties (2nd ed.), 1992.

2. Buxbaum E.: Fundamentals of Protein Structure and Function, 2007.

3. Nölting B.: Protein Folding Kinetics: Biophysical Methods (2nd ed.), 2006.

4. Nelson D. L., Cox M. M.: Lehninger Principles of Biochemistry (4th ed.), 2004.

5. Whitford D.: Proteins: Structure and Function, 2011.

6. Kessel A., Ben-Tal N.: Introduction to Proteins: Structure, Function, and Motion, 2011.

Otázky k predmetu Proteíny, štruktúra a funkcia Katedra...

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