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BMS2062 > PART V: PROTEIN STRUCTURE AND FUNCTION > Flashcards

PART V: PROTEIN STRUCTURE AND FUNCTION Flashcards

1
Q

What is structural biology?

A
  • the determination and analysis of the 3D structures of biological macromolecules
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2
Q

What does the structure/molecular details of a macromolecule (protein and/or polynucleotide) allow us to understand?

A
  • Chemistry behind the molecular mechanism or role in the cell
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3
Q

What are 7 areas that structural biology can take part in?

A
  • Drug action
  • Vitamins and minerals
  • Immune system
  • toxins and poisons
  • Molecular basis of disease
  • blood clotting
  • Viruses
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4
Q

What two things must be submitted to the PDB?

A
  • Final structure AND experimental diffraction data
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5
Q

What two key things are available from accessing the PDBID?

A
  • The 3D x,y,z coordinates of all the atoms in a macromolecular structure
  • the DATA used to determine the structure
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6
Q

In the PDB information file, will the more complex proteins have multiple chain identifiers?

A
  • YES
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7
Q

In the PDB information file, which structures is the B-factor important in?

A
  • Only meaningful in structures determined using X-ray diffraction
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8
Q

what is the B factor an indication of ?

A
  • B factor is an indication of the mobility of an atom (individual atom)
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9
Q

High B factor= protein (atoms) that are _____

A

Highly mobile

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10
Q

Are atoms on the surface of a protein generally quite mobile?

A
  • YES
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11
Q

Are atoms buried in the core of the protein generally quite mobile?

A
  • NO

- Hardly moving at all

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12
Q

What can be the case if part of the molecule of interest has high B factors?

A
  • the position of the atoms is NOT WELL DETERMINED

- So best to take caution as it absorbs ERRORS

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13
Q

If looking at a protein structure in terms of the B factor areas, what do the red and blue areas mean respectively?

A
  • Red= HOT for high B values

- Blue= COLD for low B values

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14
Q

What type of resolution method for protein visualisation makes up the majority of proteins on PDB?

A
  • X ray crystallography
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15
Q

Out of X ray crystallography, NMR and CryoEM, which one is the most up and coming?

A
  • CryoEM
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16
Q

What are the 10 common questions asked when solving a protein strucutre?

A
  1. Where are the alpha helices and beta sheets?
  2. What residues form the hydrophobic core?
  3. What is the distance between the two positions?
  4. What are the backbone angles?
  5. Is the protein MULTIMERIC?
  6. Where are the conserved residues?
  7. What is the surface electrostatic potential?
  8. Do two proteins have SIMILAR strucutres?
  9. Where is the active site?
  10. How does the protein/RNA/DNA function?
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17
Q

What are the 8 steps in Protein structure determination by X-ray crystallography?

A
  1. Express/purify the protein
  2. Crystallize the protein
  3. Collect diffraction data
  4. Determine the space group, unit cell dimensions, and number/symmetry of molecules per unit cell
  5. Solve the “phase” problem
  6. Calculate an electron density map
  7. build a molecular model to fit the electron density map
  8. REFINE the model
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18
Q

In X ray crysallography, what is 50% of the crystal made up of?

A
  • 50% made up of solvent
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19
Q

What are the general steps for obtaining samples for structure determination?

A
  • Clone the gene and INSERT into E.coli Expression Plasmid
  • grow E.coli to express the protein
  • Extract and purify the protein (multiple steps)
  • EITHER CRYSTALLIZE PROTEIN FOR X RAY DIFFRACTION OR DISSOLVE PROTEIN IN BUFFER (at high []) FOR NMR
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20
Q

What is the key to a crystals use for diffraction experiments?

A
  • It is made of repeating units –> repeating in 3 dimensions
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21
Q

Why do we need crystals for X ray crystallography?

A
  • Electrons within single molecule will scatter X rays, however we are unable to measure scattering…
  • Crystal contains REPEATING pattern of molecules –> provides a way of AMPLIFYING the signal of the scattered X rays.
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22
Q

What is involved in the X-ray diffraction pattern method?

A
  • Number of diffractions images are recorded as the crystal is rotated
  • INTENSITY of each crystal is measured
  • Each reflection is given a unique INDEX
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23
Q

What does each black spot in the diffraction pattern represent?

A
  • X-ray reflection that results from the X-rays scattering from electrons about the protein in the crystal
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24
Q

In X-ray crystallography, what are X-rays scattered by?

A
  • The electrons in the crystal
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25
Q

Can we measure the phase angle directly?

A
  • NO
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26
Q

In X-ray crystallography, what is required for each reflection before structure determination can proceed?

A
  • A phase angle
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27
Q

In X ray crystallography, once the reflections are indexed, what happens next?

A
  • Intensity is measured and phase angle determined for each reflection –> then can calculate where the electrons are in molecule–> then displayed as a 3-D contour map
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28
Q

What is an issue with X-ray crystallography in terms of the focusing and what solves this?

A
  • X rays are hard to focus –> bc. machine uses same wavelength (1 angstrom= 1*10E-10 meters) as X rays so resolution can’t be determined
  • Crystal structure ‘solves’ this as they are used to ‘mathematically focus’ the X rays
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29
Q

What does 1 angstrom = ?

A
  • 1*10E-10 Meters
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30
Q

What are 4 reasons why we use X rays?

A
  1. Will never be possible to visualise X rays using visible light even with the most powerful microscopes
  2. For an object to be seen ,its size must be at least HALF of its wavelength of the light being used to see it
  3. Visible light has a wavelength MUCH LONGER than the distance b/w atoms thus USELESS to see molecules
  4. X rays have a wavelength on the order of bond length, which must be used to visualise molecules
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31
Q

What are 4 typical bonds in macromolecules?

A
  • C-C: 1.54A
  • C-N: 1.47A
  • C-O: 1.43A
  • C-S: 1.82A
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32
Q

What does a smaller value correspond to in terms of Angstroms?

A
  • The smaller the number, the HIGHER the resolution
    e. g. 3A is worse resolution than 2A
  • An even smaller number means that there is sampling MORE OFTEN thus more acctrate
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33
Q

What value does a HIGH resolution correspond to?

A
  • Better than 2A
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34
Q

What does an ULTRA HIGH resolution correspond to?

A
  • Better than 1A
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35
Q

In terms of the accuracy of the protein model on PDB, what is it useful looking up?

A
  • The electron density map
  • Will show you how accurate the protein structure is
  • Can get parts of a protein that are not as accurate in structure as others
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36
Q

What are the key 3 methods for analysing proteins?

A
  • X ray
  • NMR
  • Electron Microscopy (CryoEM)
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37
Q

What are the SUPER basic steps for structure determination using X-ray crystallography?

A
  • Crystal
  • Data collection
  • Diffraction
  • Electron density
  • Structure
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38
Q

In NMR what units is the frequency given in on the X and Y axis?

A
  • Ppm
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39
Q

What are 3 things that NMR can be used for?

A
  • To study STRUCTRE and DYNAMICS + detecting molecular interactions
  • To study peptides, proteins, oligonucleotides and carbohydrates
  • Used to SOLVE the structure of biological molecules in solution (up to 25kDa in size EASILY but also up to 100KDa)
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40
Q

Where is the NMR signal derived from?

A
  • The radiofrequency signals of magnetically SUSCEPTIBLE nuclei -> rotate (precess) around the magnetic field
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41
Q

In NMR, what are the nuclei we observe in the study of proteins?

A
  • 1H, 13C, 15N
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42
Q

Out of 1H, 13C and 15N, which one is naturally abundant, and which other two must be prepared in an isotopically rich sample?

A
  • 1H is NAUTURALLY ABUNDANT

- 13C and 15N must be observed by preparing an isotopically rich sample

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43
Q

In the NMR experiment, if there is a small molecule that is spinning FAST, does the signal last for a short or long time?

A
  • Signal lasts for a LONG time
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44
Q

In the NMR experiment, if there is a large molecule that is spinning SLOW, does the signal last for a long or short amount of time?

A

If there is LARGE molecule that is spinning SLOW –> signal decays FAST so signal lasts for SHORT time (gone before you can measure it) -> this is the limitation of the NMR method

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45
Q

Why can NMR only be used to measure small proteins?

A
  • Because the larger molecules spin SLOW –> this means the frequency will last for a SHORT time (i.e. decay quickly) before it can be measured
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46
Q

In NMR, what does rf mean?

A
  • Rf= Radiofrequency pulse
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47
Q

What is Bo?

A

-The constant, homogeneous magnetic field used to polarize spins, creating magnetization

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48
Q

What effect does the rf pulse have in NMR?

A
  • Purtubing the net magnetization so it’s no longer aligned with Bo. –> System then returns back to equilibrium
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49
Q

in the context of NMR, which aa has NO NH side chain?

A
  • Proline
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50
Q

In a 2D spectra of NMR, what are protons that are correlated rise to?

A
  • they rise to a CROSSPEAK
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51
Q

In a 2D NOESY spectra (NMR), what is the intesnity of the crosspeak proportional to?

A
  • Proportional to how close the pair of protons are
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52
Q

In 2D NMR (NOESY), what do you have to first work out to then be able to determine the structure of a protein?

A
  • First must work out which pair of protons give rise to a NOESY crosspeak
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53
Q

In 2D NMR (NOESY), if two protons are less than 5A apart, what must be the structural explanation for that in terms of the protein?

A
  • There must be a fold in the protein structure
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54
Q

What does TOCSY do in terms of 2D NMR?

A
  • It is another type of experiment that is a CORRELATION SPECTRUM
  • Spots whenever there are COVALENTLY attached protons
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55
Q

Are NOESY and TOCSY for 2D NMR used in isolation or combination?

A
  • Used in combination to deduce which peaks correspond to which nuclei in the protein sequence
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56
Q

What is a limitation of 2D 1H NMR?

A
  • Because of the overlap, it is difficult to assign the spectrum and UNAMBIGUOUSLY identify and measure NOE constraints –> then more sophisticated strategy is required
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57
Q

What is multidimensional heteronuclear NMR?

A
  • When a protein is prepared with isotopic labels and 2D and 3D NMR experiments are conducted–> Allow through-bond connections and NOES to be visualised and measured
  • There are SPOTS at the intersection of 3 different fragments –> thus can ASSIGN spectrum (which spot belongs to which aa)
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58
Q

Can multidimensional heteronuclear NMR allow the structure to be derived using the same principles of using NOE and torsional angle constraints?

A
  • YES
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59
Q

What sized protein is Multidimensional heteronuclear NMR employed for?

A
  • Any LARGER protein (but still up to 100kDa)
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60
Q

What are the steps in protein strucutre determination in NMR spectroscopy?

A
  • Express the protein and enrich in stable isotopes (15N, 13C)
  • Optimize sample conditions (high conc., soluble)
  • Assign the 1H, 15N, 13C signals in spectra
  • Collect spectra to identify PAIRS OF ATOMS THAT ARE CLOSE IN SPACE
  • Use distance information to calculate a family of structures
  • Iterate through previous steps to refine the structural ensemble (i.e. if the computer comes up with the same answer repeatedly, then you can say you have the same structure)
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61
Q

What is the requirement for the sample preparation with X-ray and NMR respectively?

A

-X ray: Need to crystallize, NMR: Need to dissolve at high concentration

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62
Q

What is the requirement for the data collection with X-ray and NMR respectively?

A
  • Fast (Minutes to hours) and Slow (days to weeks)
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63
Q

What is the requirement for the data analysis with X-ray and NMR respectively?

A

Solving the phase problem can be slow (otherwise the process for rest is fast for X ray), NMR is relatively SLOW (increasingly automated)

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64
Q

What is the requirement for the result with X-ray and NMR respectively?

A
  • Single strucutre

- Ensemble of structures

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65
Q

What is the requirement for the best resolution with X-ray and NMR respectively?

A
  • Atomic detail (X ray)

- Generally lower resolution than X ray (NMR)

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66
Q

Is there a size limitation with X ray and NMR respectively?

A
  • No size limitation (X-ray)

- Must be <40KDa (routinely)

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67
Q

What other information obtainable does X ray and NMR contain respectively?

A
  • X-ray has binding site information that is available sometimes
  • NMR has binding site info, dynamics, equilibrium, kinetics, chemical processes
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68
Q

Which technique would you try first to determine the structure of a protein? (out of X-ray and NMR in general)

A
  • X-ray because the resolution is much higher than NMR
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69
Q

What was the HSQC experiment used to study (also what does it stand for) ?

A
  • Heteronuclear Single Quantum Coherence (HSQC) exp. is an example of 2D heteronuclear exp. which is used OFTEN to study protein interactions
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70
Q

Where does the HSQC experiment show signals?

A
  • Shows signals at 1H and 15N frequencies of each N-H in the protein (i.e. one for each amino acid backbone N-H)
  • Signal for every covalently attached N and Proton
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71
Q

What is the HSQC experiment like for the protein?-

A

A “fingerprint”

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72
Q

What is an example of how the HSQC experiment can be used?

A
  • Determining which amino acids of the protein RTP (replication terminator protein) are involved in binding its target DNA
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73
Q

Does the HSQC experiment work for every aa?

A
  • NO

- Every aa EXCEPT for proline

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74
Q

In the HSQC titration experiment (with the RTP protein), what did some NH crosspeaks moving indicate?

A
  • Indicated that the electronic environment of the proton or nitrogen had been PERTUBED due to DNA binding
  • Interpreted as being DIERCTLY involved in the binding or indirectly pertubed due to conformational change.
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75
Q

What concentration does the molecule need to be in solution for NMR?

A
  • Approx. 200microM
76
Q

What must happen to the molecule being studied for heteronuclear experiments to be carried out?

A
  • It must be isotopically labelled
77
Q

Can NMR be used for characterising molecular motion and molecular interactions?

A
  • YES
78
Q

What can binding curves be used to determine?

A
  • The number of binding sites and the affinity of each
79
Q

What are the 4 examples of non-covalent interactions?

A
  • Van der Waals interactions
  • Hydrogen bonds
  • Electrostatic interactions
  • Hydrophobic interactions
    e. g. Ion channel -toxin interaction, protein-protein interaction, receptor-ligand interaction
80
Q

What type of interactions do almost all drugs form?

A

-Non covalent interactions e.g. Kinase inhibitor interactions

81
Q

What is the equilibrium dissociation constant formula?

A
  • Kd= [P][L] /[PL] (i.e. ratio of bound to unbound at equilibrium)
82
Q

When considering the equation P +L = PL, which side will a tightly bound ligand be towards?

A
  • The PL side
83
Q

What units are Kd in?

A
  • Moles
84
Q

When considering Kd, if the ratio is 1/1000, then what does that equal?

A

= 10^-3M (=1mM)

85
Q

When considering Kd, if the ratio is 1/1 000 000, then what does that equal?

A

= 10^-6M (=1microM)

86
Q

What is Kd called?

A
  • the equilibrium dissociation constant
87
Q

What does a LOW Kd mean?

A
  • LOW Kd= TIGHT BINDING or HIGH AFFINITY

- bc. less of free P and L and more of PL

88
Q

What does a HIGH Kd mean?

A
  • HIGH Kd= LOW affinity
89
Q

When considering Kd, what is the strength of binding when it equals: 1nM, 1micoM, 1mM and 1pM?

A 
1nM= TIGHT 
1microM= moderate 
1mM= VERY WEAK 
1pM= VERY TIGHT
90
Q

Which level of Kd do we usually aim for when developing a drug in terms of binding affintiy?

A
  • Low nanomolar (nM) Kd
91
Q

What is the equation for the fraction of P (protein) bound?

A
  • [L]/Kd + [L]
92
Q

Why does the concentration of the protein have to be small when producing binding curves?

A
  • Because otherwise we cannot assume that the [] of free ligand is the same as the [] added
93
Q

When [L]= Kd, what is the fraction of P bound?

A
  • 0.5
94
Q

In the binding curves for proteins, do we have much more or much less of ligand bound?

A
  • Much more ligand than protein
  • Set up exp. so we know what the total no. of ligand is at each point and assume only tiny part is bound to the same protein –> so can assume it’s close to the same total [] of the free ligand
95
Q

What must the concentration of [P]t be 100x lower than in protein binding curves?

A
  • must be 100x lower than Kd
96
Q

What are the units for the fraction of P bound equation when looking at protein binding curves?

A
  • nM
97
Q

How do we know if the interacton measured through protein interactions is strong enough to be physiologically relevant if the Kd of H.pylori chemorecepotr TIpC to natural ligand lactate is 0.1mM?

A
  • The answer is WEAK binding BUT it is STILL physiologcially relevant–> bc. the conc. of lactate in the stomach is 1-10mM so there would be a high proportion of binding.
98
Q

What can binding curves reveal?

A
  • The number of binding sites

- 2 sites means 2 ligand molecules per protein molecule

99
Q

How do van der walls interactions arise?

A
  • Due to a combination of short range attractive forces and short range repulsive forces that occur between ELECTRICALLY NEUTRAL GROUPS
100
Q

Are van der waals interactions dynamic?

A
  • YES
101
Q

In van der waals forces, when are the atoms said to be in van der waals contact?

A
  • When the van der waals attraction EXACTLY balances the repulsive force
102
Q

In van der waals interactions, as the two nuclei draw closer together, what do their electron clouds begin to do?

A
  • Electron clouds begin to repel each other
103
Q

What do the attractive forces in Van der waals interactions originate from?

A
  • Short lived imbalances in electron distribution of an atom generating a temporary dipole
104
Q

Are Van der waals interactions weak or strong?

A
  • WEAK approx, 1kcal/mol
105
Q

In van der waals interactons, what can happen if the shapes of the two molecules match (like in ligand binding to protein)?

A
  • Additive forces makes van der waals interactions significant to the binding energy when numerous atoms in one molecule simultaneously come close to many atoms of the other molecule.
106
Q

What is a H bond in general terms?

A
  • Interaction between a polarized D-H bond (D is H donor) and the polarized non bonding orbitals of an acceptor (A)
107
Q

What is the optimal arrangement for a hydrogen bond?

A
  • D-H-A optimal binding is in a head to tail manner –> bending causes ENERGY LOSSES
108
Q

What does bending cause in H bond structure?

A
  • Energy losses which explains why the range of delta G is from 0-4kj/mol
109
Q

What law is the electrostatic interactions force given byh?

A
  • coulumbs law
110
Q

With electrostatic interactions, is the interaction of charges stronger within the protein globule than in water?

A
  • YES
111
Q

What do hydrophobic interactions arise from?

A
  • From the system attempting to achieve the grestest thermodynamic stability by MINIMISING the number of ordered water molecules to surround hydrophobic portions of the solute molecules.
112
Q

What is the Kd relarted to the sum of?

A
  • Related to the sum of all of the contributing non-colvalent interactions
113
Q

Can the atoms rotate around a peptide bond?

A
  • NO

- but can rotate around a bond that links the alpha Carbon -N and AlphaC- C

114
Q

What does each aa residue have?

A
  • 2 single bonds in the backbone

- Several single bonds in the side chain

115
Q

Protein structures are ______ dynamic?

A
  • Intrinsically dynamic
116
Q

In the thermodynamic hypothesis, what is the thermodynamics of protein folding depicted as?

A
  • A free energy funnel
117
Q

What is an example of two proteins that breaks the “one sequence, one fold” paradigm?

A
  • Ltn (Chemokine Lymphotactin)

- Prions

118
Q

In general, how do chemokines like Ltn attract leukocytes to the site of infection?

A
  • They bind to GPCRs in target leukocytes
  • this activates INTEGRINS and induces remodelling of leukocytes’ actin cytoskeleton, ARRESTS rolling of cells and promotes their TRANSMIGRATION from the blood through the endothelium, towards site of infection.
119
Q

What is Ltn10 missing?

A
  • The first 2 C residues
120
Q

Which member of the chemokine family is Ltn10 and why is it part of this family?

A
  • The C-chemokine family (only member)

- It is missing the first two disulfide bonds that are conserved in ALL OTHER CHEMOKINES

121
Q

What does the monomeric form the chemokine do?

A
  • binds to the receptor to induce leukocyte TRANSMIGRATION through the endothelium
122
Q

What are GAGS (in terms of chemokine lecture)?

A
  • glycosaminoglycans–> long linear polysaccharides that coat the surface of vascular endothelial cells
123
Q

What do chemokines secreted from injured tissue into the vasculature, adhere to, and what form of the chemokine (mono or dimeric)?

A
  • Adhere to the vasular endothelial cells by interacting with GAGs
  • Dimeric form of the chemokine binds to GAGs with HIGH AFFINITY –> promotes dimerisation
124
Q

What are the two distinct structures that Ltn adopts when in equilibrium?

A
  • Ltn10 (monomer)

- Ltn40 (dimer)

125
Q

What does Ltn10 (monomer) contribute to ?

A
  • Receptor activation
126
Q

What does Ltn40 (dimer) contribute to?

A
  • Binds to GAG (glycosaminoglycans binding)
127
Q

what are the main structures that Ltn10 contains?

A
  • Monomeric three stranded beta sheet and carbodyl terminal alpha helix
128
Q

What are the main structures that Ltn40 contains?

A
  • Dimeric all beta sheet arrangement
129
Q

Which temperature and salt conc. (in general) is Ltn10 and Ltn40 stable at respectively?

A
  • Stable at LOW temp (Ltn10) with HIGH SALT and stable at HIGH temp (Ltn40) with LOW SALT
130
Q

Are prions proteins?

A
  • Yes
131
Q

What do prions and viruses have in common?

A
  • Prions (like viruses) are infectious agents that are not cells
  • Prions also rely on the host cell to provide the machinery for their replication
132
Q

What was the first studies human disease caused by prions?

A
  • Kuru (from ritual cannabalism)
133
Q

What is prion short for?

A
  • Proteinaceous infectious particle
134
Q

How does CJD arise (prion disease)?

A
  • Spontaneous mutation in the prion protein gene –> death after 1 year
135
Q

What is GSS disease?

A
  • Hereditary dementia resulting from mutation in gene encoding prion protein
136
Q

Why are prion-based neurological diseases caled spongiform encephalopathies?

A
  • Bc. there are large fluid filled holes where neurons have died
137
Q

What are fibrils observed in infected brain tissues composed of?

A
  • Prion molecules in their MISFOLDED CONFORMATION
138
Q

What is likely a core structure of protease-resistant fibrils?

A
  • beta helixes
139
Q

In prion diseases, which component is thought to cause disease?

A
  • the fibrils
140
Q

What is a template to promote the misfolding protein in terms of prion diseases?

A
  • The beta -helix

- It hold the entire fibril together

141
Q

What is PrPc and where is it found?

A
  • A NORMAL glycoslyated cell surface protein which has an unknown physiological conformation
  • Found on the surface of nerve cells in the brain
142
Q

Is the PrPc protein essential?

A
  • NO

- Mice lacking the prion gene are healthy

143
Q

What is PrPsc?

A
  • the isoform of PrPc and has been identified as the INFECTIOUS AGENT
  • Has a MISFOLDED CONFORMATION
144
Q

Are PrPsc and PrPc thought to be chemically identical?

A
  • YES
  • They only differ in their folding
  • Helix A in PrPc folds into a beta helix in PrPsc
145
Q

How is it believed that PrPsc converts PrPc into being like itself?

A
  • Prion gnee mutations may LOWER THE ACTIVATION energy of conversion
  • this increases the Pr that conversion will happen within the average lifespan
146
Q

What is a way apart from mutation, that PrPsc can be acquired?

A
  • through the consumption of food or inoculation
147
Q

Why does the species barrier exist for prions?

A
  • bc. a certain degree of sequence identity/similarity between the host PrPc and the acquired PrPsc is REQUIRED for the interaction and catalysis to occur
148
Q

Why do intrinsic mechanisms exist against prions?

A
  • Bc. they are the hosts own proteins
  • They are NOT poisonous
  • No antibody mediated immune response to prions ever been recorded probably due to negative selection for B and T cells
149
Q

What are three reasons as to why prions are difficult to detect using conventional methods?

A
  • there are no genes, so can’t use PCR
  • Antibodies to them are NOT produced, so can’t use serology
  • Are in brain tissue and not in any of the easily accessible body fluids (blood or urine)
150
Q

What is a single pass membrane protein made up of?

A
  • Hydrophobic alpha helices
151
Q

What is a multi pass membrane protein made up of?

A
  • Amphipathic alpha helices
152
Q

What are membrane beta barrels made up of?

A
  • Multiple amphipathic Beta strands
153
Q

What are the 3 different ways membrane proteins can span hydrophobic lipid membranes?

A
  1. Single pass membrane protein
  2. Multipass membrane protein
  3. Membrane beta barrels
154
Q

What properties of side chains do non polar aa have and which side chains do they face?

A
  • Hydrophobic properties of side chains

- Face the FA side chains

155
Q

What properties of side chains do polar and charged aa have (acidic, basic, uncharged polar) an which side chains do they face?

A
  • hydrophillic properties

- Face the water

156
Q

What are the properties of the main chain of amino acids?

A
  • There are still polar properties of the amine and carbonyl (carboxyllic acid will turn into a carbonyl)
  • No longer charged BUT do prefer to make H-bonds
157
Q

Why is it important to know the properties of the main chain?

A
  • The polar parts of the aa form SECONDARY STRUCTURES by H-bonds
  • Therefore they SPAN using secondary structure
158
Q

In multi pass-alpha helices, does each helix tend to be amphipathic?

A
  • YES
159
Q

What does amphipathic mean?

A
  • Having BOTH hydrophillic and hydrophobic regions (one side of alpha helix, hydrophobic and the other hydrophillic)
160
Q

What does the hydrophobic side of the amphipathic alpha helix (normally non polar residues) interact with?

A
  • FAs of phospholipids (FA chains)
161
Q

What does the hydrophillic side of the amphipathic alpha helix (normally polar or charged residues) interact with?

A
  • With other proteins or with water channels
162
Q

Do all the resiudues that are interacting with FAs in a multipass amphipathic alpha helix need to be strictly hydrophobic?

A
  • YES all of them must be STRICLY hydrophobic
163
Q

Do all the residues that are interacting with the proteins or water channels in a multipass amphipathic alpha helix need to be strictly hydrophillic?

A
  • NO

- For example Phe and Ala are NOT hydrophillic however are present in the hydrophillic section of the alpha helix

164
Q

If we look down (superior) on a multipass alpha helix (amphipathic) which two things to we see?

A
  • One side interacts with the FAs

- One side interacts with EITHER other helix side chains OR with water

165
Q

From looking at the aa sequence of a protein, can you predict that it might be an alpha helix interacting with the membrane in a multipass situation?

A
  • NO

- You can’t be certain so must look at the structure

166
Q

What are transmembrane beta barrels, what are they made up of , and what is their polar/non polar arrangement?

A
  • Multiple beta strands
  • Amphipathic beta strands
  • Alternates with polar and non polar strands
167
Q

Why can’t you get an integrals membrane protein that has only 1 beta strand?

A
  • If there is one beta strand going through the membrane, it would be completely surrounded by FAs (first strand would be pointing towards FAs and so on)
  • Definition of a beta strand is that you can’t have a single beta strand in isolation because it MUST MAINTAIN interactions with another strand
168
Q

What is the definition of a beta strand?

A

No beta strand in isolation because it MUST maintain interactions with another strand

169
Q

What is light microscopy limited by?

A
  • 0.2mcm

- Limited by the wavelength of light and the lenses

170
Q

What is good about electron microscopy?

A
  • It has much smaller beams than atoms
171
Q

What is SP cryo-EM?

A
  • Single Particle cryo-electron microscopy

- AKA cryo-EM or cryo-TM

172
Q

What is cryo-EM good for looking at?

A

-Good for looking at big proteins or protein/DNA complexes

173
Q

What is one of the best resolutions obtained in cryo-EM?

A
  • 4.5A
174
Q

What is the requirement for the sample prep for cryo-EM?

A
  • LOW conc. and pure
175
Q

What is the data collection speed like for cryo-EM?

A
  • SLw (days-weeks) –> (like NMR)
176
Q

What is involved in the data analysis process for cryo-EM? (i.e. is phasing required and how quick is the data analysis process)

A
  • No phasing required

- Relatively slow (increasingly automated)–> like NMR

177
Q

What is the result of cryo-EM?

A
  • Single electron density map
178
Q

What is the best resolution of cryo-EM?

A
  • Variable

- Low resolution (blobby) thorugh to atomic resolution

179
Q

What is the size limitation for cryo-EM?

A
  • > 100kDa to MASSIVE!!
180
Q

What other information is available with cryo-EM?

A
  • Different combinations observed that may be excluded by crystallisation
181
Q

What are the 3 main steps in Cryo-EM?

A
  1. CRYO: Freeze the sample
  2. CRYO-EM Place the grid into a TEM (transmission electron microscopy) that has a cryostage
  3. SINGLE PARTICLE ANALYSIS: Collect data; enough images to get 1000’s of particels –> “ensemble of single particles”
182
Q

In cryo-EM are we looking at the DIRECT protein in the images?

A
  • NO
  • Looking at the reflection (shaddow) cast by the proteins in different orientations (contain the 3D info compressed onto a 2D molecule)
183
Q

What is orientation bias in cryo-EM?

A
  • Where one protein will sit in the same conformation (orientation) and many pictures will be taken of it in this SAME orientation (particle may only want to land in ONE orientation)
184
Q

What is the limit to resolution in cryo-EM?

A
  • Images–> number and quality of particles
  • Equipment–> beam intensity, direct electron detectors, etc.
  • Expertise
  • NOT enough particles/orientation bias
  • Heterogeneity of the sample (particle could be in disordered state OR there could be too many conformations)
185
Q

In order to get a 3A resolution structure of one particular state of protein, you look at the grid and there are two different conformations of the structure…How many particles would be needed to collect if in general there is 1 000 000 particles to get a structure?

A
  • 2 000 000 (2 million)
186
Q

What is SP cryo-EM good for?

A
  • INTEGRAL MEMBRANE PROTEINS (solubilised in detergent, embedded in nanodisks, embedded in lipososmes)
  • Proteins with different confomations/assembly states
  • things that do NOT crystallise
187
Q

What is SP-cryo-EM NOT good for?

A
  • Membrane proteins embedded in CELLS (bc. they have to put an artificial membrane in)
  • TINY TINY proteins <50kDa
  • Phase plate technology influencing the minimun size of the protein
  • Disordered proteins

BMS2062 (7 decks)

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