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Protein Structure and Analysis > NMR > Flashcards

NMR Flashcards

1
Q

Main strengths of NMR

A

Can study proteins at the amino acid level
Very sensitive to chemical environment
Versatile

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2
Q

NMR allows _-specific investigation of proteins

A

Amino acid

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3
Q

The position of peaks indicates

A

Chemical environment

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4
Q

What does even intensity and line width indicate?

A

Lack of motion

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5
Q

Mass limit for 3/4 structure detrmination in solution NMR

A

< 60 kDa
Needs to be small

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6
Q

Molecular mass limit for 3/4 structure determination in solid state NMR.

A

Not limited by molecular mass, in principle, but signal number and overlap constitute significant practical obstacles

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7
Q

True or false:
Solution NMR can be used above 60 kDa

A

True, just not for structure determination

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8
Q

Do you need crystals for solution NMR?

A

No

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9
Q

What is TROSY?

A

A trick to allow the study of larger systems
Can also use deuteration

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10
Q

What materials can be studied using solid state NMR

A

Dry samples, hydrated solids, sedimented soluble assemblies etc.
Doesn’t have to be soluble or crystal

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11
Q

Is NMR useful for proteins with disordered/flexible regions?

A

Yes

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12
Q

Magnetic nuclei have non-zero _ quantum numbers

A

Spin

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13
Q

Which nuclei have a spin quantum number of 0?

A

Nuclei with an even mass number and an even charge number have a spin quantum number of zero.
Like C-12

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14
Q

A magnetic nucleus oriented with (parallel to) the external field has _ energy than a nucleus oriented against (antiparallel to) the field.

A

Lower

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15
Q

What is B or Bo?

A

External magnetic field

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16
Q

Applying pulses of electromagnetic radiation at frequencies that _ the energy gaps allows observation of transitions that produce NMR signals.

A

Precisely match

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17
Q

Resolution (peak separation) increases _ with B0

A

Linearly

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18
Q

Sensitivity increases with_

A

Bo^3/2
Distinction of signals from background noise (ratio)

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19
Q

Is NMR sensitive?

A

It’s relatively insensitive, because it is quite low energy

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20
Q

What is the result of using bigger proteins for NMR?

A

Bigger protein = more nuclei = more peaks = more peak overlap
Bigger protein = broader peaks

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21
Q

To take reading of elements we often incorporate more desirable _ at the desired _.

A

Isotopes, abundance

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22
Q

How to incorporate N-15 into a system

A

You can grow cells in a medium where there is only one isotopically labelled source of nitrogen, such as 15-NH3Cl. Same applies for C13.

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23
Q

Why is it good that Nitrogen-15 has a larger range of ppms?

A

Peaks can be spread out more.
Nitrogen is especially useful because there are fewer of them in a protein

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24
Q

Rule of thumb for choosing isotopes

A
  • < 25 kDa 13C + 15N;
  • > 25 kDa 13C + 15N + 2H
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25
Why use 2-H?
To reduce peak widths
26
Signals at the intersection of 2 or more frequencies indicate a _ between nuclei
Correlation
27
How to incorporate D into a sample?
Leave in heavy water
28
How to interpret a 1H-15N HSQC?
Each dot comes from the correlation of an H with the N that it is bonded to. Pairs of peaks indicate pairs of hydrogens, same N value different H value
29
Gyromagnetic ratio values for isotopes
Constant for a particular isotope Differ between isotopes
30
What is chemical shift?
The effect of surrounding electrons on magnetic nuclei Peak positions in the NMR spectra reflect different functional groups
31
Scalar coupling or J coupling
Through bond interaction between magnetic nuclei
32
Dipolar coupling
Through space interaction between magnetic nuclei
33
Sequence specific resonance assignment:
* Determine 2o and 3o structure of proteins * Study protein interactions * Study protein stability, protein dynamics, protein folding etc
34
Why do nuclei of different isotopes resonate at different frequencies within the radiofrequency-microwave region of the EMS?
Because it reflects the different gyromagnetic ration (g) values of the nuclei e.g. in a 9.4 T magnetic field, 1H resonates at 400 MHz, 13C at 100 MHz, 15N at 40 MHz
35
NMR experiments comprise a sequence of pulses of _ radiation
rf
36
What is the resonant frequency?
Where a signal appears in the spectrum
37
What does the resonant frequency depend on?
The gyromagnetic ratio and the magnetic field strength For a particular nucleus depends on its chemical environment
38
If the protein is not folded you loose peak _
Separation
39
What are Hα?
Protons on the α Cs in the backbone
40
What is (1-σ)?
The screening constant
41
True or False: Bo is always different
True, always slightly different. No two magnets are the same
42
Why do we measure by chemical shift rather than Bo?
To remove dependence on the changing value of Bo. Instead it uses a reference
43
Protein folding results in conformation-dependent chemical shift dispersion, what does this do to the spectrum?
permits assignment of individual peaks in a spectrum to individual nuclei in a protein In favourable cases, this assignment can be made for most of the magnetic nuclei in a protein This can separate two Hs attached to the same carbon due to their position in space
44
Each amino acid produces signals with _ chemical shifts
Characteristic
45
How can you quantify affinity?
By fitting chemical shift changes or peak intensity changes
46
How much of a sample should I use?
0.3-0.5 ml of 0.02 mM-0.5 mM protein as rule of thumb ideally between 90 and 100% pure
47
What is the best kind of buffer to use?
One with lower ionic strength, Carefully controlled pH (exchange rate increases logarithmically above 2.6)
48
What can cause changes in the chemical environment?
o changes in solution conditions o intrinsic dynamics o molecular interactions o folding/unfolding o mutation
49
What does self-association do to signals?
Broadens almost all of them
50
What does conformational exchange do to signals?
Broadens only the signals of affected residues
51
What additional methods can be used to distinguish between self-association and conformational exchange?
* SEC-MALS * DLS * AUC * SAXS
52
The number of peaks can reveal _ processes
Dynamic
53
What can we determine if there are fewer peaks than expected?
That the amino acids undergo ms-ms conformational exchange
54
Why can we not see dynamics in crystalised proteins?
Because it pushes a protein to a single energy minimum
55
What do structural restraints include?
distance-, angle-, and bond orientation-based restraints
56
Advantages of Protein structure determination using solution NMR
o spectra reflect conformational heterogeneity e.g. conformational fluctuation or >1 major conformation o can study molecules in solution (including in cells)
57
Disadvantages of protein structure determination using solution NMR
* it is not a molecular imaging technique * the wavelengths involved are too long
58
Workflow for protein structure determination using NMR
* isotope labelled protein preparation * NMR data collection * Signal assignments * simulated annealing with NMR-based restraints * validation
59
What is simulated annealing?
Figure out the locations in the amino acid sequence of the secondary (2∘) structure elements (mainly a-helices and b-strands) Determine the tertiary (3∘) structure (includes the spatial relationships between the secondary structure elements)
60
Structural restraint can be from what kinds of NMR data?
Dipolar coupling - NOEs Scalar coupling - Coupling constants Chemical shift - 2* chemical shifts 1H/2H exchange - hydrogen bonds Paramagnetism - relaxation enhancement
61
What is scalar or J coupling?
Nuclei are coupled via bonding electrons, must be linked by a small number of bonds. It indicates which peaks in an NMR spectrum are from atoms covalently bonded to each other
62
The strength of scalar/J coupling reflects the _ around chemical bonds
Angles dihedral angles / torsional angles
63
J >/= 9 Hz indicates an HN group in a _
b-strand
64
J
a-helix
65
Secondary chemical shift
In a folded protein, each amino acid of the same type will have a slightly different chemical shift.
66
Chemical shifts for a-helix
ave. δCa 3.09 ±1.00 ppm, ave. δCb –0.38 ± 1.00 ppm
67
Chemical shifts for a b-sheet
ave. δCa –1.48 ±1.23 ppm, ave δCb 2.16 ± 1.91 ppm
68
What is dipolar coupling?
Magnetic field generated by nucleus j at site of nucleus k, and vice versa.
69
Why is dipolar coupling so important foe protons?
* protons stick out * protons are numerous * protons are sensitive (have big g) * Each 1H can sense other 1Hs up to about 6 Å (0.6 nm) away
70
True or false: Nuclear Overhauser Effects (NOEs) can be inter-molecular
True
71
Closer protons = _ NOE peak
Greater
72
Why is dipolar coupling so important for structure determination?
Dipolar coupling is important here, because it depends on nuclei being close in space, irrespective of whether they are close in the amino acid sequence of the protein
73
How do we determine tertiary structure from NOEs?
We enter them (100s-1000s) into an algorithm along with other structural constraints. Algorithm finds the minimum energy 3D protein structure compatible with all the NOEs.
74
What does precise mean?
Close together results
75
What does accurate mean?
Answers are close to the target
76

Protein Structure and Analysis (9 decks)

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