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Imperial Y2 - Protein Science > Alfonse de Simone - Protein Dynamics by NMR > Flashcards

Alfonse de Simone - Protein Dynamics by NMR Flashcards

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1
Q

Pros and cons of Biomolecular NMR?

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2
Q

What are the six main parameters of an NMR spectra?

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3
Q

Outline how a 1D NMR spectra is recorded

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4
Q

What is the Free induction decay (FID)? What do we need to do to our FID collected from our sample to create our NMR spectra?

A

FID refers to the perturbations/changes that occur in the magnetic field as the spin return to their ground state

The FID is collected in the time dimension - converted into the frequency dimension using the Fourier transform

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5
Q

What are the two main pieces of information obtained from the FID?

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6
Q

When two peaks have a very similar FID and the peaks fuse, what is an interesting phenomenon that is observed?

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7
Q

How can we create another time dimension when running 2D NMR?

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In order to move from 1D to 2D we need to add another time dimension – this is not actually possible (can just make a new time dimension) so instead we create a fictitious time dimension known as the indirect time

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8
Q

Outline how 2D NMR is performed?

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9
Q

What time domain in 2D NMR is manipulated?

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10
Q

By manipulating the T1 and T2 in 2D NMR, what do we end up with?

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11
Q

What is a HSQC spectra?

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The HSQC (Heteronuclear Single Quantum Coherence) experiment is used to determine proton-carbon/nitrogen single bond correlations

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12
Q

How is heteronuclear HSQC NMR performed?

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Note - The reason why we excite the 1H first is that this nucleus is more sensitive because of the highest gyromagnetic ratio.

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13
Q

What is this INEPT transfer?

A

INEPT transfer - involves a combination of pulses between Proton and heteronuclear atom that allows for transfer of energy (“polarization”) through the J-coupling (Bonds) - don’t need to know the detail

Basically, allows us to decipher which N and H+ are linked

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14
Q

What atoms are normally used for Heteronuclear NMR? What problems may arise and how do we tackle them?

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15
Q

Why is switching from a 1D NMR spectra to a 2D useful?

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16
Q

In a 1H-15N HSQC spectra, what N-H pairs are we mainly looking at?

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17
Q

In a 1H-13C HSQC spectra, what C-H pairs are we mainly looking at?

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18
Q

How are 3D NMR experiments performed?

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19
Q

What technique is used to analyze 3D spectra?

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20
Q

What is a HNCO 3D NMR spectra?

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21
Q

What is a HN(CA)CO 3D NMR spectra?

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22
Q

What do HN(CO)CACB and HNCACB 3D NMR spectra tell us?

A

Similar process as carbonyl carbon assignments but this time it is performed with the alpha carbon and Beta carbon of residues i and i-1

Normally combine HNCACB and HN(CO)CACB

HN(CO)CACB - links N-H to CA and CB of residue i-1

HNCACB - Links N-H to CA and CB of residue i

Able to assign alpha carbon and Beta carbon in relation to a N-H peak

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23
Q

What do HNCA and HNcoCA 3D NMR spectra tell us?

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24
Q

What is the main takeaway message from the popular 3D experiments performed on proteins? What can we do with all this information?

A

Takeaway - Using these techniques you can work your way through the protein backbone and assign the residues

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25
Q

When all residues in the protein have been assigned using 3D NMR, what in-silico technique is used to predict the structure

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26
Q

What does the image attached illustrate? How does Simulated Annealing NMR structure prediction differ to normally NMR structure prediction?

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27
Q

How is NMR used to study weak Protein-Protein Interactions?

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28
Q

Why is X-ray crystallography not good at studying IDPs?

A

Powerful techniques such as X-ray crystallography are inherently limited in the study of IDPs.

  1. Firstly, IDPs are virtually impossible to crystallise.
  2. Secondly, even by crystallizing an IDP under particular conditions, the resulting structure would represent one of the infinite conformations that the protein adopts in solution.

Solution –> NMR

But…

Use of traditional (e.g. NOE based) NMR would result ineffective and new methods must be developed.

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29
Q

What are the two defining characterisitcs that allow us to identify IDPs in an NMR spectra?

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30
Q

Why does NMR excite so many professors?

A
  1. NMR is a technique to study protein structure, dynamics and interactions

2. Not limited by the requirement of crystallizing samples

  1. It is suitable for weak and dynamical protein interactions
  2. Used in solution and solid-state and can therefore offer a variety of tools to study soluble and insoluble (membrane embedded or fibrillar states) proteins
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31
Q

From a conceptual standpoint how do proteins attain Biological Activity?

Think structure + dynamics

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32
Q

How are we able to break down the native state free-energy of a protein?

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33
Q

How do protein-protein interactions networks highlight the importance of protein dynamics?

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34
Q

Example of protein dynamic importance - thermophilic enzyme?

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35
Q

What are the different timescale dynamics that are examined using NMR?

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36
Q

When examining nanosecond dynamics, what are we interested in?

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37
Q

NMR Recap - When a nucleus is placed into a external magnetic field (Spins & net magnetic moment)?

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38
Q

NMR recap - What happens to the net magnetic moment when we hit the sample with a RF pulse at 90o?

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39
Q

What happens to the transverse magentization after the pulse? What does the lab and rotating frame refer to?

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40
Q

What are the two types of relaxation that are taking place when spins return to their ground state after excitaiton?

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41
Q

What is the correlation between peak width and R2?

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42
Q

What are the 4 main steps in experiments used to study Dynamics via NMR Relaxation?

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43
Q

Outline the way in which dynamic measurements - R1 & R2 are made?

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44
Q

Explain how values for R1 or R2 and obtained by using tau (τ)

A

What we do is measure different HSQCs (series of 2D spectra) with different values of τ

The τ pulse sequences used make sure that the peak intensity in the NMR spectra decay in a exponentially manner

This exponential decay can the can then be used to decipher the value for R1 or R2 - for each peak examined

As shown by the image - Tau is plotted against signal intensity creating an exponential decay curve which can be modelled by the equation shown, which is dependant on R1 or R2

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45
Q

Do R1 and R2 measurements provide us residue specific information?

A

Yeahhhhh

We end up with R1 and R2 measurements for each residue

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46
Q

Apart from R1 and R2, what is the last third type of measurement in 15N relaxation?

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47
Q

What information does Heteronuclear NOE provide us? What is the principal behind it?

A

Heteronuclear NOE

The backbone 1H-15N heteronuclear NOE provides information about the motion of individual N-H bond vectors.

Usually, the hetNOE is measured in steady state mode.

The steady state hetNOE compares the signal of the z-magnetization (signal intensity) of the 15N in thermal equilibrium to the z-magnetization of the 15N at equilibrium when the 1H is saturated

- Cross-relaxation (NOE) vs. no cross-relaxation (No-NOE) –> examine the relative contribution of 1H?

When measuring we have two modes…

  1. Equilibrium Mode - steady state mode – no saturation
  2. Saturation mode - stopping the NOE between the 1H and 15N

Interpretation of results

NOEHX ~ 1 - means that the NH vector is rigid (minimal dynamics)

NOEHX << 1 - means that there is a significant amount of dynamics in the system

48
Q

How are R1, R2 and hetNOE combined into a single parameter?

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49
Q

Summary of the standard nanosecond relaxation experiments

A
  • 15N relaxation is the most popular, it infers on N-H BUT 13C and 2H relaxation is also measurable, e.g. C’-Ca

Standard relaxation experiments normally involve measuring…

  1. R1 longitudinal relaxation
  2. R2 transverse relaxation
  3. HetNOE steady-state heteronuclear NOE

All of which is combined into… S2, order parameter

50
Q

What do the following graphs tell us about ubitquitin nanosecond dynamics?

A
51
Q

What role does AMP-dependent Protein Kinase A (PKA) Ca2+ movement in muscles? What proteins does PKA interact with?

A
52
Q

What are the 3 different states that PKA is found in? How does the proteins dynamics change between the three states?

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53
Q

What does the H-X NOE data reveal about PLN when bound to PKA?

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54
Q

What processes are we examining on the sub-ms timescale? What technique are we focusing on?

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55
Q

What type of information do we get from CPMG?

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56
Q

CPMG - How does Kex influence the NMR spectra between two equally sized populations (A & B - 50/50)

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57
Q

CPMG - What does the following NMR spectra show us?

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58
Q

CPMG - How does Kex influence the NMR spectra between unevenly sized populations (A & B - 95/5)

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59
Q

What type of spectra is normally used for CPMG?

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60
Q

How is a CPMG experiment carried out?

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61
Q

What effect does manipulating CPMG pulse frequency have on R2?

A

Therefore, in these experiments, we measure a number of HSQC 2D spectra by varying the frequencies of these pulses in the gray box.

  1. At low CPMG frequency, peaks that are in conformational exchange will have high R2 value, as the apparent R2 are the sum of the intrinsic R20 of the NMR peak plus Rex that is the conformational exchange constant between states A and B.
  2. At high CPMG frequencies we have that R2 = R20. We therefore fit the curve of R2 as function of the CPMG frequency and obtain fitting values as PA, PB, Rex and Δω
62
Q

CPMG - How do we use two-site exchange fitting to obtain PA, PB, Kex and Δω values?

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63
Q

How to obtain Rex (Kex) from CPMG relaxation dispersion curves?

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64
Q

CPMG - How do we obtain PA, PB and Δω from our relaxation dispersion curve?

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65
Q

What is ribonuclease A? What structure does it have? What is the rate limiting step?

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66
Q

Using CPMG to study net energy difference and activation energy, what did the group in Yale find out about Ribonuclease A?

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67
Q

Using the CPMG approach, what did a group of researchers find out about L99A T4 Lysozyme? Population of two states? How was the structure solved?

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68
Q

CPMG - How did T4 Lysozyme mutants change the population distribution of the two states and the substrate binding?

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69
Q

What is Dihydrofolate Reductase? What reaction does it catalyze?

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70
Q

What did CPMG data reveal about Dihydrofolate Reductase

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71
Q

What did CPMG data reveal about how Dihydrofolate Reductase efficiently moved through its reaction cycle?

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72
Q

Summary of CPMG?

A
  1. CPMG is sensitive to dynamics ranging in the ms to ms timescale
  2. CPMG is most effectively used when two conformations are in intermediate exchange
  3. CPMG provides three major data: populations, Kex and Dw.
73
Q

What effect does molecular tumbling have on relaxation?

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74
Q

What is meant by saturating H when measuring HetNOE?

A

NOE transfer of polarization from one nucleus to another through space

  • Heteronuclear NOE transfer from a proton to another type of nucleus (Nitrogen)
  • When we saturate the proton – we are introducing a frequency to remove the HetNOE, so that we are just measuring the J coupling (transfer of polarization through the bonds)

Hence, HetNOE describes the relative contribution of NOE to the spectra

a) If HetNOE = 1 then NOE has no contribution in polarization transfer rigid vector (low dynamics)
b) If HetNOE << 1 NOE has a significant contribution in polarization transfer flexible vector (highly dynamic)

75
Q

Can we get negative HetNOE numbers?

A
  • Negative number extreme phenomenon but possible
  • Indication of extremely high/fast dynamics
76
Q

Are S2 values similar to the NOEs values?

A

Yes, but S2 are more precise as they include R1 and R2 information

But in some cases NOE is accepted as an accurate representation of the S2 – often occurs when we cannot keep samples in the magnet for long periods of time, resulting in a weak signal to noise (R1 and R2 – 40 spectra required relative to the 2 spectra for NOE)

77
Q

Where did the intial idea that the apo state of proteins is more dynamic than the bound state?

A

Comes from crystal structure observations - e.g. introduction of ligand can help the crystallization process and crystallization of protein + ligand tends to create a restricted number of structures

But the introduction of NMR dynamics data highlights no real correlation between dynamics and bound/unbound states of a protein

Data shows that enzyme active site when bound to the product is especially dynamic in order to facilitate product release

78
Q

Does the wild type T4 lysozyme exhibit a dual population?

A

The L99A 97% population is nearly identical to the wild type conformation and in the wild type the secondary population is not present

Lysozyme can accommodate many mutations without compromising its structure - this mutation was performed, and its skewed population distribution was just discovered by coincidence

79
Q

Using NMR, how can we assign residual secondary structure in IDPs?

A

Basically, different secondary structure environments influence the chemical shift of a paritcular atom

Having a databse of previously known secondary structures and the chemical shifts associated we are able to assign the proability of secondary structures within our own protein

80
Q

What are the three different secondary structures that δ2D identifies in IDPs?

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81
Q

How is δ2D performed?

A
82
Q

What does δ2D reveal about the structure of prion proteins?

A
83
Q

What structures are normally examined with solid state NMR and why?

A
84
Q

Why can’t large species be studied in normal NMR?

A

Remember we are working with a large number of NMR active nuclei in a protein

  1. Each nucleus produces its own local magnetic field (dipolar field)
  2. These dipoles interact with the external magnetic field (align or not align) in turn influencing the frequency required to induce a spin change (greater or smaller energy difference) –> changing chemical shift slightly – known as time dependent field fluctuations
  3. When working with small molecules that tumble quicker, these local dipolar forces will be averaged out (faster tumbling – more efficient averaging) - having no net effect on the Delta-E – high sensitivity
  4. When working with large molecules that tumble slower, the local dipolar forces will not be averaged out - having an effect on the Delta-E –> resulting in less peak separation/sensitivity
85
Q

What do these NMR spectra show us?

A
86
Q

What happens to a normal NMR spectra when we go from liquids to solids?

A
87
Q

How do we perform solid state NMR?

A
88
Q

When performing solid state NMR, what does Cross polarisation regime and INEPT regime refer to?

A
89
Q

How doe α-synuclein interact with membranes?

A
90
Q

How does α-synuclein bind to a vesicle membrane?

A
91
Q

What were some key questions that wanted to be answered about αS binding to membranes?

A
  1. Structure and Dynamics of αS bound to synaptic-like vesicles
  2. Topological properties
  3. Molecular bases for the membrane affinity
92
Q

What does the CD spectra reveal about αS?

A
93
Q

What were the three regions in αS characterized using ssNMR?

A
94
Q

How are we able to study topological properties of proteins in ssNMR?

A
95
Q

What did Paramagnetic Relaxation Enhancement (PRE) tell us about the N-terminus of αS?

A
96
Q

What did Paramagnetic Relaxation Enhancement (PRE) tell us about the C-terminus of αS?

A
97
Q

Takehome messages about IDPs and ssNMR?

A
  1. IDPs can be studied by NMR as it provides experimental observables

Chemical shifts carry key information on the secondary structure content of IDPs

  1. Membrane proteins can be effectively studied using ssNMR

It provides structure, dynamics and topology with respect to the membrane

98
Q

What is Chemical exchange saturation transfer (CEST) NMR used to study?

A

Weak macromolecular interactions are studied using chemical exchange saturation transfer (CEST)

99
Q

How is Chemical exchange saturation transfer (CEST) performed?

A
100
Q

CEST - Outline how saturating the MB N-terminal region of αS causes signal attenuation of the soluble αS N-terminal region

A
101
Q

CEST - Outline how saturating the MB C-terminal region of αS causes signal attenuation of the soluble αS C-terminal region?

A
102
Q

CEST - What does the attached image show us? What can we take away?

A
103
Q

What was CEST data able to show about α-synuclein membrane binding?

A
104
Q

Why is α-synuclein double anchor mechanism important?

A
105
Q

CEST - What does the E46K/K80E α-synuclein do?

A
106
Q

What does addition of α-synuclein into a solution containing vesicles promote?

A
107
Q

Recap - What ssNMR and CEST taught us about α-synuclein?

A
108
Q

What does α-synuclein cause pathology on the cellular level?

A
109
Q

What did a study on α-synuclein oligomers with ssNMR reveal?

A
110
Q

How can ssNMR cross-polarization regime, INEPT regime and CEST used to study membrane protein interactions

A
111
Q

How is CEST used in MRIs?

A
112
Q

How can CEST MRI be used to gauge tissue acidosis in tumours?

A
113
Q

How is gluCEST MRI used to examine strokes?

A
114
Q

How is gluCEST used to study AD and Huntington’s?

A
115
Q

CEST summary?

A
  1. Chemical Exchange Saturation Transfer provides key information from indirect measurements
  2. It has a wide range of applications, from protein-ligand to protein-membranes interactions and to medical applications in MRI

Imperial Y2 - Protein Science (4 decks)

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