NMR Flashcards

Question

What extra measurements are made in NMR relaxation for dynamics determination

Answer 1

In two-state chemical exchange, a molecule interconverts between two conformations or binding states When both states are equally populated (50% A, 50% B), the appearance of the NMR spectrum depends on the exchange rate (k e x ex ) relative to the chemical shift difference ( Δ ω Δω) between states. Slow Exchange ( k e x ≪ Δ ω k ex ≪Δω) The exchange rate is much slower than the chemical shift difference. Two distinct peaks are observed at the chemical shifts of A and B. Each peak has equal intensity (50:50 ratio). Intermediate Exchange ( k e x ≈ Δ ω k ex ≈Δω) The exchange rate is similar to the shift difference. Peaks broaden significantly and may merge into a single, broad, distorted peak. This regime is often studied with CPMG relaxation dispersion experiments to extract exchange rates. Fast Exchange ( k e x ≫ Δ ω k ex ≫Δω) The exchange is much faster than the shift difference. A single peak appears at the weighted average chemical shift: δ obs = p A δ A + p B δ B p A + p B δ obs = p A +p B p A δ A +p B δ B Since p A = p B = 50 % p A =p B =50%, the peak appears at the midpoint between A and B. Peak broadening depends on k e x k ex .

Answer 2

It reveals dynamic processes where a nucleus (e.g. an amide proton) interconverts between two (or more) chemical environments with different chemical shifts In CPMG relaxation dispersion NMR, we exploit the fact that: If a nucleus is exchanging between two (or more) chemical environments (say A and B), its (R2) relaxation rate becomes dependent on the rate and extent of exchange. By varying the frequency of refocusing pulses we can measure how the observed transverse relaxation rate, R2eff changes — this is called a dispersion curve. Why It’s Useful • Detects “invisible” excited states (low population, short-lived) • Measures protein conformational dynamics, folding intermediates, binding kinetics • Often used with 15N, 13C, or 1H NMR on backbone amides

Answer 3

CSA refers to the fact that the chemical shift of a nucleus depends on the orientation of its local electronic environment relative to the magnetic field (B₀). In other words, the magnetic shielding a nucleus experiences is direction-dependent — it's anisotropic (not the same in all directions). Why does CSA happen? Nuclei are surrounded by electrons, which create tiny local magnetic fields when exposed to B₀. These local fields shield the nucleus from B₀ to varying degrees depending on molecular orientation. For non-spherical (anisotropic) electronic environments, the amount of shielding changes with direction → leading to different chemical shifts for different orientations. Molecules tumble rapidly in solution, averaging the CSA effect. The observed chemical shift is the isotropic average. BUT: CSA still affects relaxation, especially T₂ (transverse) and T₁ (longitudinal) relaxation.

Answer 4

CEST is used to detect low-population, invisible states that are in slow exchange with a visible, higher-population state. How it works: A selective radiofrequency pulse is applied at the frequency of the invisible or minor state. If there's exchange with a major state, the saturation transfers to the major state and causes a decrease in signal intensity. By scanning the saturation frequency, you generate a CEST profile, revealing where the minor state exists and how it exchanges. DEST is similar in principle to CEST but optimized for situations where the dark state is extremely broad and essentially invisible in the NMR spectrum (e.g., large aggregates or bound states with very fast transverse relaxation) How DEST works: A broad saturation pulse is applied to the region of the spectrum where the dark state would resonate. Exchange with a visible, narrow-line free state transfers the saturation, reducing its signal. DEST curves (plots of signal attenuation vs. saturation frequency) can be analyzed to extract information about the dark state.

Answer 5

CEST detects low-population, “invisible” states (e.g., excited conformations, weakly bound ligands) that are in slow exchange with a major, visible state (usually on the milliseconds to seconds timescale). Apply a weak, selective radiofrequency (RF) saturation pulse at a specific frequency — typically where the minor state would resonate. If the minor state exchanges with the major state, the saturation is transferred to the major state over time. This reduces the signal intensity of the major state. By scanning the saturation frequency, you create a CEST profile — a plot of signal intensity vs saturation frequency. Info obtained from CEST: exchange rate, population of the minor/invisible state, chemical shift difference between states, Structural/functional info about invisible states (e.g., conformational changes, binding events) DEST is used when the minor (dark) state is extremely broad and invisible — such as aggregates, very large complexes, or strongly relaxing bound states. From fitting the DEST profile, you can determine: Exchange rates between free and dark (bound) states Bound state population Transverse relaxation rates of the dark state Binding affinity and kinetics, even if the bound state is too broad to observe directly

Answer 6

An NMR method to probe exchange of NMR visible species with excited states. If the excited state has a large MW the method is also called DEST (dark exchange saturation transfer) Based on the application of a weak saturating field (B1) at various places (offsets) on the spectrum When an NMR resonance is saturated (ie when the B1 field is applied at tis frequency : offset ~0) its intensity is diminished (dark blue) If there is exchange with another conformation that has a large MW saturation of the main observable species is observed at large offsets (light blue) This happens because saturation of the large exited state is transferred to the small NMR observable main state through chemical exchange

Answer 7

Since membrane proteins are embedded in the lipid bilayer, they cannot be directly studied in solution NMR. Therefore, the proteins need to be reconstituted into model lipid bilayers (e.g., lipid vesicles, bicelles, or lipid nanodiscs) that mimic the natural membrane environment. Solid-state NMR is uniquely suited for studying membrane proteins because it works with samples in the solid phase or non-crystalline states, making it applicable to proteins embedded in membranes or lipid bilayers. High resolution is achieved by spinning the sample rapidly (at around 10-70 kHz) to average out anisotropic interactions like dipolar couplings and chemical shift anisotropy, which typically broaden NMR peaks. Cross-polarization is used to transfer magnetization from abundant nuclei (like ¹H) to less abundant nuclei (like ¹³C or ¹⁵N), improving the sensitivity of the NMR experiment. Membrane proteins have well-defined inter-atomic distances that can be measured using dipolar couplings (through the H-H, C-H, or N-H interactions)

NMR Flashcards

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