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Protein Structure and Function: How current anesthetic practices evolved from South American blow dart hunting

PVAMU 02-25-2011 Maria Fadri-Moskwik, PhD

maria fadri

on 27 February 2011

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Transcript of Protein Structure and Function: How current anesthetic practices evolved from South American blow dart hunting

Learning outcomes Anesthesia and South American blow dart hunting neuromuscular nAChR Studying protein structure and function of the nAChR Learning outcomes
be able to name one example of how conservation of protein structure and function facilitates biomedical research (include the name disease, molecular target, and model organism)
identify three ways which researchers can determine protein structure
identify the ion channel receptor by which the neurotoxin, curare, affects the neuromuscular junction
discuss how amino acid substitutions can effect protein structure and function Experimental methods to determine protein structure
X-ray crystallography
NMR Spectroscopy
Cryo-Electron Microscopy (EM)
X-ray crystallography NMR Spectroscopy Cryo-EM relies on magnetisation of nuclei in an exterior magnetic field >>>Earth's magnetic field
requires solubilization of the proteins in deuterated solvent (dynamic)
small proteins or peptides
Nobel prizes (Van Laue, papa and son Bragg, Hodgkin, Perutz and Kendrew Structural biology
Interdisciplinary field with aspects of molecular biology, biophysics, and biochemistry
How macromolecules are organized in space
Why macromolecules have the structure they do
What happens to function if structure changes 1 melggpgaprllpplllllgtgllrasshvetrahaeerllkklfsgynkwsrpvanisdvvlvrfglsiaqlidvdeknqmmttnvwvkqewhdyklrwdpadyenvtsiripseliwr
241 rlplfytinl iipclliscl tvlvfylpse cgekitlcis vllsltvfll liteiipstslvipligeyl lftmifvtls ivitvfvlnv hhrsprthtm ptwvrrvfld ivprlllmkr
481 gvrcrsrsiq ycvprddaap eadgqaagal asrnthsael pppdqpspck ctckkepssvspsatvktrs tkappphlpl spaltravegvqyiadhlkaedtdfsvkedwkyvamvidr
601 iflwmfiivc llgtvglflp pwlagmi
Jahnichen and Unwin, 2005
4A resolution quaternary structure nAChR
(Steen Pedersen, PhD) nAChR a-subunit x-ray diffraction pattern crystal electron density map atomic model relies on X-ray diffraction to map molecules in a protein (elastic scattering)
requires crystallization of a protein (non-trivial for membrane proteins)
small molecule (<100 atoms) and macromolecule (arbitrarily large size)
Nobel prizes (Van Laue, papa and son Bragg, Hodgkin, Perutz and Kendrew resolution
restraints Methot, N et al, Biochemistry, 1994 Nicotinic AcetylCholine Receptor (nAChR)
pentameric ligand-gated ion channel
two types--neuronal (nicotine addiction), and muscular (muscle relaxant, anesthesia)
binding to ligand (acetylcholine) causes channel opening
non-selective cation channel (K+, Na+) neuron releases acetylcholine (ACh)

ACh binds to the receptor (nAChR)

nAChR opens and allows cations to enter muscle cell

muscle cell depolarizes and contracts Molecular modeling and mutational analysis
1. given primary structure (amino acid sequence) and homologous protein, computer program generates a structure

2. mutate amino acids to determine whether they are important for ligand binding or channel function

3. identify inherited mutations and determine effects channel function Pacific electric ray (Torpedo californica) can generate 45 volts of energy to subdue prey or in self defense

nAChR of the electric ray is HIGHLY HOMOLOGOUS to the human nAChR
makes this a good model system for research
allows purification of large amounts of nAChR to do biochemical and biophysical assays Protein Structure and Function:
How South American blow dart hunting gave rise to modern general anesthetic practices soluble sample

sequence assignment

side chain assignment

collection of NMR restraints

NMR structure calculation

structure validation

What happens if ACh can't bind to nAChR?

Curare competes with acetylcholine for nAChR binding and blocks muscle contraction! The race to "discover" surgical anesthesia and the Ether Dome
Crawford Long--performed surgery with ether 1842, published in 1848
Horace Wells--dentist who successfully had his own teeth removed under nitrous oxide; later public demonstration (1845) "failed" because while patient did not feel pain or wake up but struggled during procedure
William T. G Morton--anesthetized patient undergoing tumor surgery with ether

Ether Day at the Ether Dome: Oct 16, 1846 at Massachusetts General Hospital RC Hinkley, "First Operation Under Ether" Balanced anesthesia is a cocktail of drugs that in combination:
analgesic (numb pain)
sedative (consciousness)
muscle relaxant (suppress movement and reflexes)

Bodily functions (heart, lungs, brain, other organs) are closely monitored to ensure the right amount of anesthetization

The story of curare: how blow dart technology and an electric fish helped to revolutionize surgery and medicine Use of curare and its derivatives in surgery
1942, Griffiths and Johnson administered curare during surgery to lessen reflexive responses
also used to facilitate tracheal intubation
patient will require artificial respiration (diaphragm is affected) but their heart will continue to beat
side effects of curare (histamine release and hypotension) led to use of curare derivatives with fewer side effects (pancuronium, mivacurium)

two classes of neuromuscular blockers = non-depolarizing blocking agents and depolarizing blocking agent curare/tubocurarine
isolated from vines of South American (Amazonian Rainforest) plant, Chondrodendon tomentosum

applied to tip of blow darts, used in hunting
causes flaccid paralysis of voluntary muscles (muscles can't contract)
method of delivery is injection (can't pass into the blood stream from the stomach) ACTION AT THE NEUROMUSCULAR JUNCTION Step 1: Obtain lots and lots of protein Step 2: Obtain experimental data on protein structure Step 3: Confirm structural data with computational modeling or mutational analysis--refine structure primary structure
(amino acid sequence)

secondary structure
(alpha helix, beta-sheet)

tertiary structure
(atomic coordinates)

quaternary structure
(subunit assembly) Rosalind Franklin's X-ray diffraction pattern for DNA relies on X-ray diffraction to map molecules in a protein (elastic scattering)

requires crystallization of a protein (non-trivial for membrane proteins)

small molecule (<100 atoms) and macromolecule (arbitrarily large size)

Nobel prizes (Van Laue, papa and son Bragg, Hodgkin, Perutz and Kendrew U. Arizona Biochem462 Molecular modeling allows you to make predictions Schapiro, M et al, Structural Biology 2002; Acetylcholine in the nAChR binding pocket ab initio calculations (small molecules)

threading programs (need sequence of amino acids and homologous protein structure

computationally "mutate" proteins to determine binding energies X-ray crystallography and DNA Case study A patient, a toddler presents with the following symptoms
severe muscle weakness
feeding and respiratory problems
delays in the ability to sit, crawl, and walk

Blood chemistry and electrolyte tests come back normal
Cognitive tests come back normal
Cardiovascular tests come back normal
Electromyography indicates decreased muscle activity in response to stimulus
Nerve conduction tests come back normal Inherited mutations of the nAChR and disease
Biochemistry assays allow you to test predictions based on molecular modeling and structural studies competitive binding assay with different mutants identifies:

which amino acids are important for binding the ligand

what sort of interactions are required for ligand binding

Wilcoxson I et al, JBC 2002 autosomal dominant nocturnal frontal lobe epilepsy (neuronal nAChR)
congenital myasthenia syndromes (neuromuscular nAChR)
arthrogryposis multiplex congenita type Escobar (fetal neuromuscular nAChR)
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