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PHD > BCH 184 NMR > Flashcards

BCH 184 NMR Flashcards

1
Q

Describe how a nucleus’s magnetic moment is related to its spin quantum number and gyromagnetic ratio.

A

By combining the magnetic moment µ=γP with the Spin Angular Moment P= h[bar] x √(I(I+1)) we obtain μ= h[bar]γ×√(I(I+1)) where I is the spin quantum number

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2
Q

Identify the NMR behavior (inactive/Active) and spin quantum number of a nucleus based on its atomic number and spin quantum number.

A

If mass number is even and atomic number is even I = 0
Mass number even atomic odd spin is an integer
If mass number is odd spin number is a 1/2 multiple

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3
Q

Describe how a nucleus’s larmor frequency is related to its gyromagnetic ratio and magnetic field.

A

Larmor frequency ω=−γB_0 where B is an external magnetic field, so the resonance frequency v_L=ω/2π=(−γB_0)/2π

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4
Q

Describe the Zeeman effect. Know how to relate the energy difference between two states of a spin 1/2 with its larmor frequency or gyromagnetic ratio

A

The Zeeman effect is the splitting of the energy levels of the spin states in the presence of a magnetic field
E=−μ_Z B_0 =−mγh[bar]B_0
∆E=γh[bar]B_0

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5
Q

Describe the limiting factors for the sensitivity of NMR method.

A

The ratio of nuclei in the a state and the b state, the more equal the ratio the less sensitive the NMR method is

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6
Q

Describe the factors that affect the macroscopic (net) magnetization

A

-the magnitude of the static magnetic field, B_0
-The identity of the nucleus()
-Temperature (T)
-the number of nuclei N (sample concentration)

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7
Q

Describe the difference between rotational frame and laboratory fame in NMR measurements

A

In the rotational frame the coordinate system rotates about the z-axis at the same frequency as the reference rotating component B_1 making apear static, in the lab from the coordinate system is locked in place

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8
Q

What are the most important Spin 1/2 Nuclei in biochemistry?

A

1H
13C
15N
31P

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9
Q

Know the definition of chemical shifts. In chemical shift unit, what does ppm mean?

A

δ=10^6 ((v−v_ref ))/v_ref

This makes the definition of the chemical shift in parts per million, in 800 MHz mag field 10ppm will be 8000Hz

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10
Q

Know that chemical shift is tensor and is magnetic field dependent. Why can we represent it is using a value under aqueous conditions?

A

In an aqueous solution the chemical shift is averaged out by how fast the molecules tumble around so only a single isotropic chemical shift value is observed

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11
Q

How does shielding effect impact the chemical shift? Know the different sources of the chemical shifts origins.

A

Inductive effect, ring effect

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12
Q

How does inductive effect impact chemical shift?

A

Electronegative elements pull electron density away from the hydrogen nucleus, thereby decreasing the magnitude of the shielding field

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13
Q

What is the “ring” effect, how does it affect the chemical shifts of interior and exterior protons respectively?

A

The ring effect is from the de localized electrons of ring working with and against the B0 field, making the internal protons will experience a weaker magnetic field and the external ones will experience a stronger magnetic field

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14
Q

Which two sections does Fourier 1D NMR experiment consist of?

A

The B0 static field and the B1 RF pulse field

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15
Q
  1. What is the definition of the B_1
A

It is the pulse RF magnetic field applied on the xy plane

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16
Q

What is FID? After Fourier transformation, what information does a FID give?

A

Free induction decay is when you use a strong magnetic signal that you decrease over time. Chemical shifts of a single class of nuclei are detected

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17
Q

Know how to draw a pattern of NMR peaks for a nucleus under fast exchange, intermediate, and slow exchange regime on an NMR time scale. Which two ways can be used to diagnose the NMR exchange regime of an NMR signal?

A

Slow exchange limits have two distinct sharp peaks, as the exchange limit increases to intermediate the peaks will widen and overlap into one broad hump, then as they become fast into one large sharp peak

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18
Q

In the 1D 1H NMR spectrum of a proteins, what are the features for an unfolded protein? What are the features for a folded protein?

A

Folded protein generally gives an NMR signal of 8.7 pp, or large for NH backbones in water and signals less than 0.5 ppm for CH3
Unfolded protein if featured wi a narrow range of backbone NH (8.1-8.5ppm) in water and CH3 (0.8-2ppm)

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19
Q

What is the mechanism of spin-spin (J) coupling? What is the difference between the origins of spin-spin coupling and dipolar coupling?

A

When two nuclear spins become coupled due to the influence of bonding electrons on the magnetic field running between the two nuclei. Spin spin coupling occurs when the nuclie are connected by covalent bonds, dipolar do not need to be physically connected by a bond.

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20
Q

What is the “Roof effect”?

A

The inner peaks become larger than the out peak

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21
Q

Under what condition will strong coupling be considered?

A

Δδ<10 J

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22
Q

Know how to draw the peak splitting patterns for nuclei with strong coupling.

A

The split peaks merge

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23
Q

What is Karplus curve? What structural information can be derived from the measurement of (_^3)J_HNHα ?

A

The karplus curve is the dependence of J coupling on the torsion angle, can be used to determine the backbone angle of protein structures

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24
Q

What interaction (_^3hJ_NC coupling be used to identify?

A

The presence and strength of N-H..O hydrogen bonds

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25
Q

Know the definition of spin decoupling. What are the homonuclear coupling and heteronuclear coupling, respectively? What advantages does this technique have?

A

Spin decoupling is when the averaging of some nucleic spins cease to influence adjecent nuclei, causing the observed spin-spin splitting to be reduced
Homonuclear decoupling is when the same kind of nuclie are being observed decoupling, heteronuclear ias when they are different nuclei

26
Q
  1. What sample condition is normally used to measure residual dipolar coupling?
A

Weakly aligned mediums

27
Q

What is the NMR relaxation? What is Nuclear Overhauser Effect (NOE)?

A

NOE is the transfer of nuclear spin polarization from one nuclear spin population to another via cross relaxation

28
Q

Know how to draw the energy diagram for transient NOE.

A

Like dis

29
Q

What is the definition of the NOE enhancement factor? Know the equations for transient NOE enhancement. Know the relationship between NOE enhancement and the distance of nuclei

A

NOE enhancement factor is defined as Intensity in enhanced spectrum - intensity in reference spectrum / intensity in reference spectrum
For transient NOE:
η(τ_m )=2σ_IS τ_m

30
Q

What does “Pulse sequence” mean? List three components of a pulse sequence. Able to describe/draw the pulse sequence for spin-echo experiment. Can explain how the magnetization of different nuclei (with different Chemical shifts) become refocused in the end.

A

A pulse sequence describes a set of radio frequency (RF) pulses applied to a sample to produce a specific form of NMR signal.
Its three elements are Pulse, Delay and data acquisition

31
Q

Know the definition of the B0 and B1 field.

A

B0 is the static magnetic field, B1 is the magnetic field generated by an RF pulse

32
Q

What are the definition of NMR T1 and T2 relaxations? What is the major mechanism for NMR T1 and T2 relaxation, respectively?

A

T1 relaxation, also known as Spin-Lattice relaxation and Longitudinal relaxation is the excited spins return to the ground state.
T2 relaxation also known as Spin-Spin Relaxation and Transverse relaxation is the loss of spin coherence in the x-y plane

33
Q

Know the correlation between line broadening and T2 relaxation time

A

Long T2 relaxation time means narrow spectral lines yielding high resolution spectrum

34
Q

What does a spin-echo pulse sequence do?

A

Refocuses magnetizations of nuclei regardless of their chemical shift difference

35
Q

Understand the general scheme of 2D NMR?

A

Two dimensional NMR is recorded as a function of two time variables, t1 and t2, and the resulting data is Fourier transformed twice to yield a spectrum which is a function of two frequency variables.

36
Q

What is the advantage of 2D NMR over 1D NMR?

A

2D NMR helps overcome the assignment problem, where several overlapping signals can be difficult to assign to resonances uniquely

37
Q

What respective NMR information does the diagonal peaks and cross peaks of 2D 1H COSY provide?

A

Diagonal peaks indicate covalently linked 1H nuclei, Cross peaks provide connectivity and J coupling constants between them

38
Q

What respective NMR information does the diagonal peaks and cross peaks of 2D 1H TOCSY provide? In comparison with 2D 1H COSY, what additional information could 2D 1H TOCSY provide?

A

With long enough mixing time, correlations between different nuclie, even with J coupling so small that their correlations can not be observed by 2D COSY, in the same spin system can be established

39
Q

What does “Spin System” mean?

A

A group of nuclei connected by J-couplings

40
Q

What respective NMR information does the diagonal peaks and cross peaks of 2D 1H NOESY provide?

A

Information on specific nuclei groupings, because of the 1/r^6 dependence we generally see no NOESY peaks for protons further than 5-6 Angstroms apart

41
Q

What information does the intensity of the NOESY cross peaks provide?

A

Strong Peaks suggest protons are in close proximity, weak peaks that they are farther apart

42
Q

In a transient NOE experiment, what is the drawback if the mixing time τ_m is too short or long?

A

If time it too short, the NOE effect cannot build up, if it is too long the peaks will disappear due to Spin Diffusion and the system relaxing back to equilibrium

43
Q

What is spin diffusion?

A

A situation wherein the individual nuclear spins undergo continuous exchange of energy

44
Q

What is the primary advantage of multi-nuclear, multidimensional NMR over 2D 1H NMR?

A

While adding a layer of complexity, labeling allows unique identification of sites in the molecule using pairs of frequencies and less spectral overlap

45
Q

List two stable isotopes that are often needed to be enriched in the protein sample in a multinuclear NMR experiment. Why do they need to be enriched?

A

13C and 15N, enhancement if needed to obtain full assignment, particularly for proteins with over 100 amino acids

46
Q

List four parts that a 2D 1H-15N HSQC pulse sequence is composed of?

A

-INEPT
-Magnetization of 15N frequency
-Reverse INEPT: magnetization transfer from 15N back to 1H
-Detection of FID by the receiver during T2 (decoupling pulse is applied to 15N)

47
Q

What does INEPT stand for? (BCH 184)

A

Insensitive Nuclear Enhancement by Polarization Transfer

48
Q

What information does a 2D 1H015N HSQC NMR spectrum provide?

A

Structural information about the amide backbone

49
Q

What information do the 3D HNCACB and CBA(CO)NH experiments provide? Describe how to achieve protein backbone chemical shift assignment using the combination of these two NMR spectra.

A

HNCACB provides intra and Inter-residue Connectivity, CBCA(CO)NH provides only inter-residue connectivity, Combining them can connect the amino acids sequentially by “Walking” along the poly-peptide backbone chain

50
Q

Describe what NMR experiments are generally used for chemical shift assignment of protein side chains.

A

An HCCH-TOCSY to collect all 13C and 1H nuclei in the side chain of each residue, (H)C(CO)NH to get the 13 C backbone, H(CCO)NH to get the 1H backbones, and HBHA(CO)NH to connect the 1H_alpha and 1H_beta Backbones

51
Q

Why do you need to apply a decoupling pulse on 15N during T2? What will the peak pattern become if there is no such decoupling applied?

A

The will become doublets instead of single peaks

52
Q

Discuss the pros and cons of NMR spectroscopy vs X-ray method in macromolecular structure determination.

A

X ray methods have short data collection and structural calculation process and can be used on large proteins, however they require being crystallized and therefore are analyzed in non-physiological conditions.
NMR can be done in aqueous solution, much closer to physiological environment which is useful since many proteins can be difficult to crystallize, but they need smaller targets (usually lower than 20kD) and often require isotope labeling

53
Q

List the most commonly used experimental restraints for NMR structure calculation

A

1H-1H NOE:
Dihedral Angle:
Hydrogen Bond:
Residual Dipolar Coupling (RDC):

54
Q

How do you obtain 1H-1H NOE information?

A

obtained from NOESY experiments

55
Q

How do you obtain Dihedral Angle information?

A

Derived from experimentally measured spin-spin couplings or predicted from the secondary chemical shift information

56
Q

How do you obtain Hydrogen Bond information?

A

Derived from the experimentally observed 3h_J_NC couplings or inferred from the NOE restraints or secondary structure information

57
Q

How do you obtain Residual Dipolar Coupling (RDC) information?

A

measured from a difference in the coupling constant between aligned and unaligned media

58
Q

What information is taken as input for automated NOE assignment?

A

Amino acid sequence, sequence-specific assignments, and NOESY cross peak positions and volumes

59
Q

What are the input requirements for a successful automated NOE assignment?

A

Completeness of chemical shift assignments and Quality of NOE peaks

60
Q

What is the output information from the automated NOE assignment?

A

3D Structures

61
Q

Briefly explain what is simulated annealing. What kind of structure calculation issue does a simulated annealing protocol help to overcome?

A

A starting structure is heated to a high temperature in a simulation, followed by cooling steps, during the steps the starting structure can evolve towards energetically favorable final structures derived from the constraints. The process helps overcome the local energy minimal

62
Q

List the five parameters that are commonly used for evaluating the quality of NMR structures

A

-Molecular energies
-Number of experimental constraints
-Root-Mean-Square Deviation (RMSD) of structural ensemble
-Violation of constraints/restraints
-Comparison to known structures (PROCHECK)

PHD (7 decks)

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