Alfonse de Simone - Protein Dynamics by NMR Flashcards

Question

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

Answer 1

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.

Answer 2

1. NMR is a technique to study protein **structure, dynamics and interactions** **2. Not limited** by the requirement of **crystallizing samples** 3. It is **suitable for weak and dynamical protein interactions** 4. 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

Answer 3

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

Answer 4

Yeahhhhh We end up with R1 and R2 measurements for each residue

Answer 5

**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

Answer 6

- 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

Answer 7

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

Answer 8

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.

Answer 9

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)

Answer 10

- Negative number extreme phenomenon but possible - Indication of extremely high/fast dynamics

Answer 11

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)

Answer 12

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**

Answer 13

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**

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

1. IDPs can be studied by NMR as it provides experimental observables Chemical shifts carry key information on the secondary structure content of IDPs 2. Membrane proteins can be effectively studied using ssNMR It provides structure, dynamics and topology with respect to the membrane

Answer 18

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

Answer 19

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

Alfonse de Simone - Protein Dynamics by NMR Flashcards

(115 cards)