Structure and Design of Biomolecules

81 flashcards · Biology · by Ajay Pradhan

1. Visible light, which is electromagnetic radiation with wavelengths of 400-700 nm cannot produce an image of individual atoms in protein molecules, in which bonded atoms are only about 0.15 nm apart.

2. True or false? X-rays are diffracted by even the smallest molecules. [True]

3. True or false? X-ray analysis of proteins can always resolve the hydrogen atoms. [False]

4. Why is it not possible to produce a focused image of a single molecules even though individual atoms diffract x-rays?

   X-rays cannot be focused by lenses - need to measure the directions and strengths (intensities) of the diffracted x-rays A single molecule is a very weak scatterer of x-rays- most of the x-rays will pass through a single molecule without being diffracted, so the beams are too weak to be detected. A crystal of a protein contains many ordered molecules in identical orientations, so each molecule diffracts identically, and the diffracted beams for all molecules augment each other to produce strong…

5. A crystal is an object with translational symmetry

6. What is the desired purity of protein for x-ray crystallgraphy?

   better than 95% purity if possible

7. What are the prerequisites for protein cyrstallisation?

   Need a few mg of purified protein Must be pure (better than 95% purity if possible) Must be homogeneous - protein isoforms and micro-heterogeneity can be very damaging to crystal growth Need high concentration of protein The protein must be stable throughout the experiment

8. What are the main things that affect protein solubility?

   pH Salts Polar solvents Temperature

9. How does pH affect protein solubility?

   pH changes some groups from neutral to charged or from charged to neutral. This alters surface charges and interaction with water.

10. What are the differences between salting in and salting out?

    Salting in is the addition of salt to increase protein solubility, while salting out is the addition of salt to decrease protein solubility.

11. What do polar solvents such as PEG (polyetheleneglycol) do?

    Polar solvents soak up water

12. How does temperature affect protein solubility?

    Thermodynamic factors influence solubility

13. Protein solubility is affected by adding precipitating agent

14. How do you take protein to supersaturation in a controlled way?

    by adding precipitant by drying out the drop by exchanging the buffer (dialysis)

15. What are the different methods for protein crystallization?

    Hanging drop Sandwich drop Sitting drop

16. What does it mean if you see a clear drop during protein crystallization? What can you do to get protein crystals?

    It means the precipitating agent or the protein concentration is too low. We can wait, or use seeding or use cat whiskers (take a whisker, touch a crystal and streak through a drop)

17. What do you do in seeding? (Protein crystallography)

    You crush up existing crystals and dilute and include in setups. You can take a whisker, touch a crystal and streak through a drop too

18. What are the possible reasons for just getting precipitates during protein crystallization?

    Protein could have been denatured Micro-heterogeneity could be disrupting crystal packing Need to improve preparation and purificaiton Other protein sources to find more likely candidates

19. What are the solution for protein crystallization if the protein is too flexible?

    Use additives like metals or inhibitors to increase stability Mutagenesis and other sources to increase stability Break up and target sub-domains

20. How do you solve the problem of getting too many micro-crystals?

    Micro-seeding (adding crushed up and diluted crystal seeds) Streak seeding (cat-whisker and streaking through new drop) (transfer of crystal nuclei)

21. Crystals can be damaged by freezing, what kind of cryo-protectant are used before freezing?

    Typical glycerol or PEG400

22. A synchrotron is used for collecting x-ray diffraction as the source of x-rays.

23. Lattice planes are the planes passing through the lattice points

24. What is indexing?

    Assigning indices to planes

25. In screw axes, mnm_nmn​ means what?

    mnm_nmn​ means translation of n/mn/mn/m of the unit cell and then rotation of 360/m

26. 5-fold rotational symmetry is not possible in crystals

27. Inversion and mirror symmetry are not possible in protein crystals

28. In crystallography only 2, 3, 4 and 6 fold rotational symmetry are possible.

    Because rotational symmetry must also be consistent with translational symmetry

29. How many primitive unit cells are possible in proteins?

    7

30. The main tool to add and subtract waves is?

    Fourier Transform

31. Fourier transform converts time domain to frequency domain.

32. How does a point (atom) scatter a wave (x-ray)?

    A point scatters a wave with equal amplitude in all direction

33. What are the different types of crystal systems?

    Triclinic Monoclinic Orthorhombic Tetragonal Cubic Hexagonal Trigonal

34. Charged particles are accelerated by electric fields.

35. What happens when an electron interacts with an electromagnetic wave?

    It oscillates at the same frequency of the wave. Then generates electromagnetic radiation with the same frequency and 180 degree out of phase. This is called scattering

36. What are the names of two types of scattering?

    Rayleigh scattering Mie scattering

37. During scattering by an atom, what is the phase difference between the incident waves and the scattered waves?

    pi radians or 180 degrees

38. What happens when the scattering angle gets wider?

    As scattering angle gets wider, atoms lose scattering power

39. There are 14 Bravias lattices

40. I

    Body centered crystal

41. P

    Primitive crystal

42. F

    Face-centered crystal

43. C

    Side-centered crystal

44. Out of a total of 230 space groups possible, only 65 are possible for proteins

45. X-rays are scattered by electrons

46. Scattering decreases with increasing angle

47. The scattering power of an atom increases with an increase in its number of electrons

48. What is anomalous scattering?

    A phase shift other than pi, resulting in a complex contribution to the scattering amplitude

49. What is the form factor of an atom?

    The dependence of amplitude on the scattering angle is called the form factor of an atom

50. Crystallography uses discrete or continuous Fourier Transform?

    Discrete

51. What is the x-ray scattering from a unit cell called?

    Structure Factor

52. What are the methods for solving the phase problem?

    1. Direct methods 2. Molecular Replacement 3. Heavy atom methods

53. How do you get the heavy metals in your protein crystal?

    soaking or co-crystallization

54. What do you add to chemically fix samples for conventional electron microscopy?

    - Karnovsky buffer - glutaraldehyde - osmium tetraoxide - Uranyl acetate -Lead citrate

55. NMR is about the interaction of magnetic fields with nuclear spins

56. The interaction of a spin with a magnetic field consists of absorbing energy from electromagnetic radiation by going from the low to the high energy state, or of emitting radiation when falling back to the low energy state.

57. NMR allows monitoring of interactions between an external magnetic field and atomic nuclei.

58. In NMR experiments, the energy is absorbed by the nuclei, potentially passed over to neighbouring nuclei, and finally emitted and detected by receiver coils.

59. What properties of proteins do NMR experiments help to study?

    Passing of energy (or magnetisation, see later) between nuclei provides a basis for the gathering of data about the three-dimensional (3D) structure of a molecule Relaxation of the nuclei towards an equilibrium is related to dynamic aspects of the molecule small modifications of the local magnetic field by charge or spin properties of neighbouring particles register the presence, and thus binding of other molecules

60. What can NMR be used to study?

    3D structure Internal dynamic behaviour Intermolecular interactions

61. For NMR we will only considers atoms with a nuclear spin of 1/2

62. What are the atoms that we are interested in looking at in NMR experiments?

    1H, 13C, 15N, 19F They all have nuclear spin of 1/2 So the hydrogen is the regular hydrogen but the carbon and nitrogen are isotopes (the rarer one)

63. Not suitable oxygen isotope is available.

    This is a very important atom.

64. True or False? Each nucleus with spin 1/2 may lead to a signal in a NMR spectrum [True]

    A one-dimensional spectrum recorded for a protein is typically very crowded and not very useful due to extensive signal overlap.

65. Why is a one-dimensional NMR spectrum recorded for a protein not very useful?

    A one-dimensional spectrum recorded for a protein is typically very crowded and not very useful due to extensive signal overlap. This is because each nucleus with spin 1/2 will lead to a signal/

66. Higher dimensional spectra contain signals described by n coordinates, where n is the number of dimensions and each coordinate is a frequency belonging to a spin ½ nucleus. As an example consider a two-dimensional (2D) spectrum: a (off-diagonal) signal has two different coordinates belonging to two nuclei; this signal indicates a magnetisation (energy) transfer between the two nuclei.

67. In NOESY spectra, every pair of hydrogens separated by a distance of less than ~5Å yields a signal. A less complex spectrum is obtained by the 15N-HSQC experiment: every HN-N moiety, e.g. from the backbone of every residue except for prolines, results in a signal (Fig. 1.3C); 15N-HSQC spectra are rapid to record and provide an efficient way to monitor structural, dynamic and binding behaviour with near-atomic resolution. This rapid spectrum is in particular very useful in drug discovery when sc…

68. Stern-Gerlach experiment was the experiment that discovery nuclear spins.

    Topic: NMR

69. What are the main components of the NMR instrument?

    magnet probe transmitter duplexer receiver computer

70. In the the formula given below, μ\muμ , S and γ\gammaγ stand for what? μ\muμ = magnetic moment S = Spin γ\gammaγ = gyromagnetic ratio

    From NMR theory

71. What does the h with the cross mean? reduced Plank's constant

72. Write the formula for energy difference. [NMR]

73. What is chemical shift?

74. How do electronegative atoms affect the magnetic field locally? [NMR]

    Electronegative atoms draw electrons away from other nuclei and thereby reduce the electronic shielding effect on these nuclei. This causes the resonance frequency of the affected nuclei to increase

75. What are the sources of chemical shift?

    Electronegative atoms reducing the electronic shielding effect Ring current effects pi electrons in the aromatic rings jumping from one ring atom to the next. Outer magnetic field B0 will induce a ring current in an aromatic ring. This current will yield a small new magnetic field B', which is opposing B0 above and below the ring plane, and reinforcing B0 in the ring plane.

76. What is the resonance condition in NMR?

    Where ω\omegaω = angular momentum γ\gammaγ = gyromagnetic ratio B = magnetic field

77. Write the formula for chemical shift

    B is the total final magnetic field, B0 is the original magnetic field and B' is the magnetic field due to the chemical shift gamma is the gyromagnetic ratio ω\omegaω is the angular momentum

78. What is the unit of chemical shift?

    ppm Unit of ppm helps to omit the factor of 106

79. What else do chemical shifts in proteins depend on besides electronegative atoms and aromatic rings?

    Residue type Local secondary structure

80. Where do the different parts of a protein lie in a 1-dimensional NMR spectra?

81. When does J-coupling occur?

    J-Coupling occurs between nuclei that are connected by a small number of chemical bonds.