# Welcome! Course Number: Course Title

NMR and MRI Membrane Biophysics Charles Holcombe and Jason Cote Overview

Basics of NMR Solution NMR Paper SSNMR Paper Review of MRI MRI and TBI Paper Diffusion Tensor Paper

Introduction to Nuclear Magnetic Resonance MagLab 700 MHz 52 mm solution state NMR system Introduction to Nuclear Magnetic

Resonance Phenomenon of NMR was discovered by Purcell and Bloch in 1948 Based on interactions of magnetic moments of nuclei (nuclear spin) with magnetic fields Odd number of protons means a non-zero spin (magnetic moment) 1H, 13C, 15N have a spin of Align parallel or antiparallel to a magnetic field (quantum numbers of +/- )

Introduction to Nuclear Magnetic Resonance dM/dt = -M x B = -M x M x B = -M x M x B = -M x - Gyromagnetic constant: nuclear magnetic moment/angular momentum - Angular frequency (2) is referred to as Larmor frequency) is referred to as Larmor frequency ) is referred to as Larmor frequency is in Hz

Faradays Law changing magnetic flux induces electromotive force in a coil Right hand rule RF Pulse in a coil perpendicular to the static magnetic field creates a secondary magnetic field at an arbitrary angle 90O is excitation pulse 180o is inversion pulse

Introduction to Nuclear Magnetic Resonance (MHz T 1 (MHz T 1 Introduction to Nuclear Magnetic Resonance If there are 2 energy states, the amount of energy required to

transition from one to the next is derived as follows: E= E= Since, , then When the frequency matches the energy between the two states, resonance occurs Example (MHz T1)) 1H: 42.58 13C: 10.71 15N: -4.32

Introduction to Nuclear Magnetic Resonance Nuclear Relaxation: Spin-spin or transverse relaxation time T2 is the decay time constant of in the transverse plane Spin-lattice or longitudinal relaxation time T1 Dipolar Relaxation Fluctuating magnetic fields from neighboring nuclei (e.g., 1H)

Chemical Shift Normalized standard resonance frequency tetramethylsilane (TMS) =() is referred to as Larmor frequency ) is referred to as Larmor frequencyref)/) is referred to as Larmor frequencyref Measured in ppm Introduction to Nuclear Magnetic Resonance Spin-Spin Coupling or J-coupling

Spectral lines are split depending on the neighboring electrons/nuclei Coupling constant J magnitude of interaction (Hz) Multiplicity is generally equal to n+1 where n is the number of neighboring nuclei Very useful in structure analysis and determination! Introduction to Nuclear Magnetic Resonance Spectral line intensity

Line intensity proportional to molar concentration of 1H nuclei In summary, we gain information on how many different types of H, the type of H, how many of each type of H, and how many neighboring H Introduction to Nuclear Magnetic Resonance 1D vs 2D NMR Spectrums (simplified)

1D: Pulse, observe, FT( time to frequency domain) and CS 2D: pulse, wait (t1), pulse, observe (t2), 2D FT and plot 3D in 2D (think topography) Types of Nuclear Magnetic Resonance Solution NMR vs Solid State NMR Chemical shift anisotropy (CSA) and dipolar coupling are averaged out due to random motion in solutions CSA and dipolar coupling dominate in solid-state meaning increased complexity

SSNMR Provides more information on the spatial orientation of the molecules since the information is not lost to random motion. Dynamic Nuclear Polarization (DNP) Papers Reviewed Solution NMR studies reveal the location of the second transmembrane domain of the human sigma1 receptor

Jose Luis Ortega-Roldan , Felipe Ossa, Nader T. Amin, , Felipe Ossa, Nader T. Amin, Jason R. Schnell Membrane Protein Structure and Dynamics from NMR Spectroscopy Mei Hong, Yuan Zhang, and Fanghao Hu Solution NMR studies reveal the location of the second

transmembrane domain of the human sigma-1 receptor Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor Sigma-1 receptor (S1R): membrane chaperone protein found in both ER and plasma membranes

Accessory protein to ion channels and receptors Voltage-gated potassium, sodium, and calcium channels; IP3 receptors, acid sensing channels, and glutamate and dopamine receptors Found in CNS and binds small molecules Opiates, antipsychotics, antidepressants, antihistamines, phencyclidine-like compounds, -adrenergic receptor ligands, cocaine, dimethyltryptamine, progesterone, and sphingosine

Potential therapeutic target Schizophrenia, Alzheimers and Parkinsons diseases, amnesia, depression, amyotrophic lateral sclerosis and addiction Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor Complete structure and relationship to the membrane is not

known Contains 2 TM domains connected by a cytosolic domain and an ERaccessible C-terminal chaperone domain Know only regions from residues (regions 91-109 and 176-194) SBDL1 and 2 (Steroid Binding Domain Like) are located within S1R ligand binding site Sequence based predictions of TM helices are not conclusive

Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor Goal is to define residues that constitute TM2 Utilized solution NMR studies of novel S1R construct (S1R(E= 35)) in which the first TM has been removed Enables description of residues within TM2 helix and secondary

structure of cytosolic domain Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor Sample Prep The S1R(E= 35) sample contained only the 36-223 residues of S1R and was confirmed by sequencing

C94A substitution was shown to have no effect on ligand binding and was introduced to prevent disulfide bonds during purification Purified protein were incubated with 1% dodecylphosphocholine (DPC) and the lipid DPPC added the sample to a q ratio of 0.1 (lipid/detergent ratio) Dodecylphosphocholine (DPC) micelles are useful as a model membrane system for solution NMR Dipalmitoylphosphatidylcholine (DPPC) is a phospholipid

Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor TROSY-HSQC spectrum 600 Mhz Solution NMR studies reveal the location of the second

transmembrane domain of the human sigma-1 receptor Far UV circular dichroism spectra similar helical content per residue and consistent with NMR chemical shifts (42% and 46% for S1R(E= 35) and S1R(cd), respectively) Solution NMR studies reveal the

location of the second transmembrane domain of the human sigma-1 receptor Chemical shift-based secondary structure analysis of S1R(E= 35)

13 C chemical shift index Solution NMR studies reveal the location of the second transmembrane domain of the human

sigma-1 receptor Dynamic properties of S1R(D35) from relaxation ratio collected at 600 MHz 1 H15N heteronuclear NOEs

Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor Chemical shift perturbations in the backbone amide 1H and 15N resonances of S1R(D35) in DPC upon addition of DPPC to a DPC:DPPC ratio of 1. The solid line indicates the sum of the absolute chemical shift perturbations (CSP) over a 7residue window, with the magnitude of the CSP for each residue (blue bars). A gray bar indicates prolines or residues for which the chemical shift assignments could not be determined. Solution NMR studies reveal the

location of the second transmembrane domain of the human sigma-1 receptor The characterized secondary structure of S1R missing only 8 residues Nterminus and the first transmembrane domain Membrane Protein Structure and Dynamics from NMR Spectroscopy

Membrane Protein Structure and Dynamics from NMR Spectroscopy Why SSNMR? Membrane proteins solubilized only in detergents and lipids (which have large molecular weights) are difficult to resolve in solution based NMR Large crystals are difficult to grow SSNMR allows membrane proteins to be studied in phospholipid

bilayers Represents biological membrane much better than micelles SSNMR gives precise information about protein orientation and dynamics Membrane Protein Structure and Dynamics from NMR Spectroscopy Two major advantages of SSNMR

Magic-angle spinning (MAS) of the samples to obtain high-resolution spectra that reflect isotropic chemical shifts and recoupled, orientation-dependent (anisotropic), dipolar couplings and chemical shift anisotropy Read, use MAS to remove anisotropy and coupling complexity and then add coupling and anisotropy back to gain some additional information that was lost Oriented Sample NMR Align lipid membranes to determine protein orientation in the phospholipid bilayer

Dipolar coupling and chemical shift become well resolved Difficult to align (either mechanically in glass plates or magnetically using liquidcrystalline media Membrane Protein Structure and Dynamics from NMR Spectroscopy Membrane Protein Structure and Dynamics from NMR Spectroscopy Magic Angle Spinning NMR

Simplify things 54.74o Membrane Protein Structure and Dynamics from NMR Spectroscopy Potassium Channels 2D and 3D MAS to assign majority of residues and mapped out chemical shift changes channel activation, inactivation and inhibition 1H-1H distance measurements and chemical shifts showed how

different scorpion toxins had different effects on channel Kaliotoxin perturbs upper region of filter, but leaves the filter in a conducting state which induces the closed confirmation of the intracellular activation gate Tetraphenylporphyrin causes a collapsed selectivity filter and an open activation gate Membrane Protein Structure and Dynamics from NMR Spectroscopy Proton Channel

M2 protein of the influenza A virus forms a tetrameric pHactivate proton channel important to virus life cycle Used MAS SSNMR, Oriented-sample NMR and solution NMR Overhausser effects used in solution NMR to determine spatial proximity 13C-2H REDOR Rotational Echo Double Resonance Very accurate distance determination between C and H 0.3 A resolution structure 2H NMR Dynamics information

H nuclei show quadrupolar splittings, which depend on the average orientation of the C-D bond in the magnetic field Membrane Protein Structure and Dynamics from NMR Spectroscopy Drug binding site in influenza M2 TM with 13 2 C- H REDOR

Membrane Protein Structure and Dynamics from NMR Spectroscopy Orientation determination with 2H SSNMR Orientation of the C-D bond in the magnetic field

Membrane Protein Structure and Dynamics from NMR Spectroscopy Conformational plasticity of M2 channel Membrane Protein Structure and Dynamics from NMR Spectroscopy GPCRs 2H SSNMR structural and dynamic changes of retinal upon light

absorption -Sheet-Rich Proteins Solution NMR high resolution structure of human VDAC Viral fusion proteins Static 31P NMR to study HIV fusion peptide (gp41) Phospholamban

Studied by OS, SS and solution NMR Different structures of PLN cytoplasmic domain indicate dependence of protein structure on lipid composition, protein/lipid ratio, protein hydration Magnetic Resonance Imaging What is Magnetic Resonance Imaging (MRI)?

MRI is a non-invasive and versatile technique that allows for the imaging of bones, organs, and tissues without ionizing radiation Application of the static or main magnetic field (B0) and Radio Frequency (RF) Pulses Uses inherent magnetic properties of human (and other animal) tissue to produce contrast Taking advantage of the abundance of 1H

Proton Imaging How does it work? Spin But it is the behavior of the averaged magnetic properties of each individual spin that matters for the resonance phenomenon How does it work?

Net Magnetization (M) How does it work? Application of RF pulses at the resonance (or Larmor) frequency results in precession of the net magnetization about B0 f0 Precession frequency Gyromagnetic Ratio B0 Strength of the main magnetic field

T1/T2 Relaxation How does it work? MR signal is the result of the precessing net magnetization inducing a current in receiver coils around the MRI machine Faradays Law of Induction Traumatic Brain Injury? (TBI)

Damage to the brain from external mechanical force Affects 1.7m annually, 52,000 deaths, over 250,000 hospitalized Deficits include impaired learning and memory, and mood disorders such as depression and anxiety Penumbr a Diffuse Axonal Injury

The Adams Diffuse Axonal Injury Classification: Grade 1: Mild diffuse axonal injury with microscopic white matter changes in cerebral cortex, corpus callosum, and brainstem Grade 2: Moderate diffuse axonal injury with gross focal lesions in the corpus

callosum Grade 3: Severe diffuse axonal injury with finding as grade 2 and additional focal lesions in the brainstem. fuse axonal injury is a clinical diagnosis Sagittal FLAIR

Axial FLAIR Can MRIs sensitivity to DAI predict injury outcomes? Diffusion Tensor Imaging Diffusion tensor imaging (DTI) comprises a group of techniques where calculated eigenvalues (1, 2, and 1, 2, and 3) and eigenvectors (1, 2, and 1, 2, and 3) are used to

create images reflecting various diffusion (water) properties of a tissue. Solving of the Stejskal-Tanner equation equation describes the signal intensity change at each voxel: Diffusion weighted to Diffusion Tensor The direction is

different for each image, resulting in a different pattern of signal loss (dark areas) due to anisotropic diffusion. Fractional Anisotropy Measuring diffusion asymmetry in a voxel

Issue of Crossing Fibers

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