Proteins

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Sept 14, 2009

GMS BI 555/755 Lecture 2.

1

Review:


Gibbs free energy, enthalpy, entropy


Work and energy storage/utilization in biological systems


Types of chemical bonding (covalent, H
-
bonding, electrostatic,
Van der Waal’s)


The hydrophobic effect


Protein microenvironments


Properties of water, acid
-
base
equilibria

Sept 14, 2009

GMS BI 555/755 Lecture 2.

2


Reading: Berg 6th ed. Chapter 2 (Supplemental: Creighton:
Proteins
)


Primary Structure


Amino acid side chains and classification


The peptide bond


Secondary structure


Peptide bond angles and rotation


Alpha helix


Beta sheet


Turns


Tertiary structure


Hydrophobic effect


Effects of solvent


Folding motifs


Protein folding problem


Molecular chaperones


Quaternary structure

GMS BI 555/755 Lecture 2: Levels of Protein Structure

Sept 14, 2009

GMS BI 555/755 Lecture 2.

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Protein primary structure

Proteins are polymers of L
-
amino
acids linked by peptide bonds

Amide bond

~60%

~40%


Amide bonds have a substantial degree of
planar character


Chemically unreactive. Hydrolysis at pH
extremes

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The peptide bond


Peptide (amide) bond very stable in solution
in the absence of a catalyst

Peptide bonds may be trans or cis, trans being
favored because there are fewer unfavorable steric
interactions

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Formation of the peptide bond


Peptide bond is an amide
bond


Very stable


Positive
Δ
H


Peptide bond formation
increases order (negative
Δ
S)


Not a spontaneous process
(becomes spontaneous when
coupled to a process such as
ATP hydrolosys)

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Glycine and alanine

A, G:


Neutral


Small R group (low
accessible surface area)


Non
-
polar


Flexible

G: achiral, most flexible AA

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Aliphatic amino acids

V,L,I,M:


Neutral


High surface area


Non
-
polar


Hydrophobic


Van der Waal’s
interactions in folded
interior


Structural units with a
variety of shapes


I side chain is chiral


V,L,I: common

M: Rare


Easily oxidized to
sulfoxide then sulfone

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Methionine oxidation

Methionine residues are susceptible to oxidation
in vivo

and during protein
workup and characterization

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Aromatic amino acids

F, Y, W:


Neutral


Very high accessible surface area

F: very non
-
polar, hydrophobic

W: rare

W, Y: responsible for 280 nm absorbance

W: Fluorescent properties

A =
ε
BC

A = absorbance

ε

= molar absorbtivity

C = concentration

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AAs with alphatic hydroxyl group

S, T:


Neutral


Polar, H
-
bonding donors or acceptors


Hydrophilic or hydrophobic


Sites of post
-
translational modification


Phosphorylation (S, T, Y)


O
-
glycosylation (
β
-
O
-
GlcNAc,
O
-
glycans)


T side chain chiral

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Cysteine


Sulfhydryl (thiol) most reactive group in proteins


Oxidation in presence of oxygen


Very nucleophilic, reactions with electrophiles


Must be alkylated (stabilized) for effective
analysis


Reactions with metal ions


Participates in disulfide bonding with other
cysteine residues. Important secondary
structure stabilizing event in proteins.


Antioxidant, precursor to glutathione

Cystine: disulfide bonded Cys residues

Cys is nucleophilic and must be alkylated for analysis (reaction
with iodoacetic acid)

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Cysteine alkylation

Fluoresceine
-
5
-
maleimide

Derivatizing groups for cys stabilization

Fluorescent alkyl groups

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Homocysteine: analog
of Cys and Met,
metabolic intermediate


Elevations of homocysteine occur
in the rare hereditary disease
homocystinuria and in the
methylene
-
tetrahydrofolate
-
reductase polymorphism genetic
traits. The latter is quite common
(about 10% of the world
population) and it is linked to an
increased incidence of thrombosis
and cardiovascular disease and
that occurs more often in people
with above minimal levels of
homocysteine (about 6 μmol/L)


Risk factor for vascular disease,
Alzheimer’s Disease


Darvesh, S., Walsh, R., and Martin, E. (2007) Homocysteine
Thiolactone and Human Cholinesterases.
Cell Mol Neurobiol

27
, 33
-
48.

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The basic amino acids

Histidine Ionization. Histidine can bind or release
protons near physiological pH.

pKa ~ 6

pKa ~ 12

R, K:


Positively charged at pH 7


Most basic protein groups (also N
-
term)

H:


Can participate in acid/base reactions at pH 7

nucleophilic

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Hydroxylysine


Biosynthesized from lysine oxidation by lysyl oxidase


Found only in animal proteins, mostly in collagen as a PTM


6
-
67 of 1000 AA residues of collagen are hydroxylysine


17
-
90% of collagen lysyl residues are hydroxylated


Hydroxylysine is typically found in triple
-
helical regions almost exclusively in the Y
positions of the repeating
-
X
-
Y
-
Gly
-

sequences in various collagens.


Embryonic tissues contain much more hydroxylysine than adult tissues.


Hydroxylation of lysyl residues in collagens prevents deposit of minerals between
fibrils


Lysine hydroxylation seems to be increased as well in some diseases, for
example, lipodermatosclerosis, osteoporosis, and osteogenesis imperfecta


Precursor to collagen crosslinking


Hydroxylated lys residues may be glycosylated

http://herkules.oulu.fi/isbn9514267990/html/x319.html

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Ornithine lactamization: ornithine is
unstable in peptide chains due to its
propensity to form 6
-
membered cyclic
lactams

Ornithine is one of the products of the action of the enzyme arginase
on L
-
arginine, creating urea. Therefore, ornithine is a central part of
the urea cycle, which allows for the disposal of excess nitrogen.


Ornithine is not an amino acid coded for by DNA, and, in that sense, is
not involved in protein synthesis. However, in mammalian non
-
hepatic
tissues, the main use of the urea cycle is in arginine biosynthesis, so
as an intermediate in metabolic processes, ornithine is quite important
(wikipedia)


Ornithine (analog of Lys, product of arginase)

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AAs with side chain carbonyls

D, E are acidic, hydrophilic

Neutral, hydrophilic

D,E:


pKa~5


Very polar


Usually charged in
proteins


Esterification
reactions possible

N,Q:


Neutral


Polar, H
-
bonding


Deamidation reactions
(protein ageing)

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Protein deamidation


Deamidation is a common post
-
translational modification


Conversion of Asn to a mixture of
Asp and isoaspartate (aka beta
-
aspartate).


Occurs to a lesser extent with Gln


Deamidation may cause loss of
protein activity


An important consideration for
recombinant protein
-
based drugs
and therapeutics


Occurs
in vivo
, especially among
proteins with long life times.


Highest frequency for Asn
-
Gly
sequences


Intermediate frequency for Asn
-
X
where X = polar (Ser, Thr, Asp)


Low frequency for Asn
-
X where X
= hydrophobic residue


Asn must be on flexible portion of
protein


Alkaline pH accelerates
deamidation


Change in protein acidity

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Proline


Cyclic imino acid


No rotaton about N
-
Cα bond


No backbone N
-
H H
-
bonding.


No resonance stabilization of amide
bond


Peptide bond more likely to be in
cis
-
conformation


Trans and Cis X
-
Pro Bonds.

The energies of these forms are relatively
balanced because steric clashes occur in both forms.

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Hydroxyproline is produced by hydroxylation of the amino acid proline by
the enzyme prolyl hydroxylase following protein synthesis (as a post
-
translational modification). The enzyme catalysed reaction takes place in
the lumen of the endoplasmic reticulum.


Hydroxyproline is a major component of the protein collagen.


Hydroxyproline and proline play key roles for collagen stability. They
permit the sharp twisting of the collagen helix. In the canonical collagen
Xaa
-
Yaa
-
Gly triad (where Xaa and Yaa are any amino acid), a proline
occupying the Yaa position is hydroxylated to give a Xaa
-
Hyp
-
Gly
sequence. This modification of the proline residue increases the stability
of the collagen triple helix.


It was initially proposed that the stabilization was due to water molecules
forming a hydrogen bonding network linking the prolyl hydroxyl groups
and the main
-
chain carbonyl groups. It was subsequently shown that
the increase in stability is primarily through stereoelectronic effects and
that hydration of the hydroxyproline residues provides little or no
additional stability.


Hydroxyproline is found in few (animal) proteins other than collagen. The
only other mammalian protein which includes hydroxyproline is elastin.
For this reason, hydroxyproline content has been used as an indicator to
determine collagen and/or gelatin amount. (wikipedia)


There are 28 types of
collagen, over 90% of the
collagen in the body are of
type I, II, III, and IV.


Collagen One
-

bone (main
component of bone)


Collagen Two
-

cartilage (main
component of cartilage)


Collagen Three
-

reticulate
(main component of reticular
fibers)


Collagen Four
-

floor


key
component of basement
membranes


Twisted, left handed helix due
to high Pro, Gly content.


Hydroxyproline

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Lesk:
Introduction to Protein Science
, chap 3, Fig. 1

Space filling amino acid side chain structures

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Codon usage and protein structure evolution

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Table of the frequency with which one amino acid is replaced by others in the
amino acid sequence of the same protein in different organisms

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Rotation of peptide bonds in a polypeptide

Dihedral angles in a polypeptide

Rotation About Bonds in a Polypeptide.

The
structure of each amino acid in a polypeptide
can be adjusted by rotation about two single
bonds. (A) Phi (φ) is the angle of rotation about
the bond between the nitrogen and the α
-
carbon
atoms, whereas psi (y) is the angle of rotation
about the bond between the α
-
carbon and the
carbonyl carbon atoms. (B) A view down the
bond between the nitrogen and the α
-
carbon
atoms, showing how φ is measured. (C) A view
down the bond between the α
-
carbon and the
carbonyl carbon atoms, showing how y is
measured.

The dihedral angles of a sequence of amino
acid residues defines the three dimensional
structure of the protein backbone

A Ramachandran Diagram Showing the Values of
φ and
Ψ
.

Not all φ and
Ψ

values are possible without
collisions between atoms. The most favorable
regions are shown in dark green; borderline regions
are shown in light green. The structure on the right is
disfavored because of steric clashes.

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Protein secondary structure: alpha helix


Structure of the α Helix.

(A) A ribbon
depiction with the α
-
carbon atoms and
side chains (green) shown. (B) A side
view of a ball
-
and
-
stick version depicts
the hydrogen bonds (dashed lines)
between NH and CO groups. (C) An
end view shows the coiled backbone
as the inside of the helix and the side
chains (green) projecting outward. (D)
A space
-
filling view of part C shows the
tightly packed interior core of the helix.


3.6 res/turn


H
-
bonding to i+4

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Proteins with high
α
-
helical content

A Largely α Helical Protein.




Ferritin, an iron
-
storage protein, is built from a bundle of α helices.

Myoglobin: first protein
structure reconstructed by X
-
ray
crystallography (Kendrew and
Perutz), proved prediction of
α
-
helix structure by Corey and
Pauling

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Protein secondary structure:
β
-
sheets

An Antiparallel β Sheet.

Adjacent β strands run in
opposite directions. Hydrogen bonds between NH and
CO groups connect each amino acid to a single amino
acid on an adjacent strand, stabilizing the structure.

A Parallel β Sheet.

Adjacent β strands run in the same
direction. Hydrogen bonds connect each amino acid on one
strand with two different amino acids on the adjacent strand.

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Protein secondary structure:
β
-
sheets

A mixed
β
-
sheet

Ribbon diagrams of twisted
β
-
sheets

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Protein secondary structure:
β
-
sheets

Loops on a Protein Surface.



A part of an antibody
molecule has surface loops (shown in red) that mediate
interactions with other molecules

Structure of a Reverse Turn.

The CO group
of residue
i

of the polypeptide chain is
hydrogen bonded to the NH group of residue
i

+ 3 to stabilize the turn

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What determines whether
a particular protein
sequence (sub
-
sequence) forms an
α
-
helix,
β
-
sheet, or a turn?


Amino acid residues have
varying propensities to be
present in secondary
structures.


α
-
helix (default),
branched R
-
groups
disfavored (V,T,I); H
-
bond
donating R
-
groups
disfavored (S, D, N)


β
-
strands more tolerant
of bulky R groups


Proline disrupts
α
-
helices
and
β
-
sheets, found in
turns.

Values diff’t in 5
th

ed

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Hydropathicity/hydrophobicity index

Kyte, J., and Doolittle, R. F. (1982)
J Mol Biol

157
, 105
-
132.


Kyte and Doolittle hydrophobicity

K&D Hydrophobicity plot for human rhodopsin

Expasy (http://ca.expasy.org)

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Prediction of protein secondary structure from AA
sequence

K&D

Chou and Fasman

Computed scale of alpha helix
forming properties for the 20 AA
based on known protein structures


Chou P.Y., Fasman G.D. Adv.
Enzym. 47:45
-
148(1978).


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Tertiary structure: the overall three dimensional fold of a
polypeptide chain

Diagram depicting the amino acid
backbone of myoglobin as a ribbon (8
helices) but no side chains

Ball and stick model showing
all myoglobin atoms but not
showing the amount of space
each occupies

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Tertiary structure of proteins: driven by the hydrophobic
effect

Distribution of Amino Acids in Myoglobin.

(A) A space
-
filling model
of myoglobin with hydrophobic amino acids shown in yellow, charged
amino acids shown in blue, and others shown in white. The surface of
the molecule has many charged amino acids, as well as some
hydrophobic amino acids. (B) A cross
-
sectional view shows that mostly
hydrophobic amino acids are found on the inside of the structure,
whereas the charged amino acids are found on the protein surface.

Figure 3.46. “Inside Out” Amino Acid
Distribution in Porin.

The outside of porin
(which contacts hydrophobic groups in
membranes) is covered largely with
hydrophobic residues, whereas the center
includes a water
-
filled channel lined with
charged and polar amino acids.

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Protein sequence motifs: structural elements (folds) found
in different proteins

Greek key motif of beta
strands


Protein motifs are three dimensional structures
(folds) found in a diversity of proteins and
protein families. Their presence may imply a
certain class of function (structural, enzymatic,
or adhesive)


Algorithms exist for predicting the presence of
motifs from the primary sequence.

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Protein folding motifs

Richardson, J. S. (1994) Introduction: protein motifs.
Faseb J

8
, 1237
-
9.

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The role of solvent in secondary/tertiary structure formation

Reduction and denaturation of ribonuclease

Anfinson, 1950s: Reduced, denatured
ribonuclease regains enzymatic activity
when urea and
β
-
mecaptoethanol are
removed by dialysis


Chaotropic (denaturing) agents
form H
-
bonds with water, disrupt
the normal structure of water,
change the energetic balance that
favors sequestering hydrophobic
sequences in interior domains. In
the absence of the entropic
driving force behind protein
folding, unfolding occurs.

SDS

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Protein folding


Proteins have the capacity to fold and become active based on the
information contained in their amino acid sequence.


Thermodynamically spontaneous


Proteins fold in buffered water;


Chaotropic agents disrupt the structure of water by participating in hydrogen
bonding. As a result, the hydrophobic driving force that makes a folded
structure energetically favorable is disrupted


Guanidine salts, urea, detergents


Proteins also denature at pH values deviating significantly from neutral.


Water miscible organic solvents are able to participate in hydrogen bonding.
Their presence also alters the thermodynamic driving force behind protein
folding. As the percent of organic solvent in a solution increases, the
tendency of proteins to unfold increases.


Heat increases molecular motion. As proteins heat they fold and unfold
rapidly. Intermolecular interactions of hydrophobic domains may cause
proteins to precipitate (cooking an egg).

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Proteins fold cooperatively, not randomly

Lodish

A current view of protein folding. Each domain of a
newly synthesized protein rapidly attains a “molten
globule” state. Subsequent folding occurs more
slowly and by multiple pathways, often involving the
help of a molecular chaperone. Some molecules may
still fail to fold correctly. These are recognized and
degraded by specific proteases.

Components of a Partially Denatured Protein Solution. In a half
-
unfolded
protein solution, half the molecules are fully folded and half are fully
unfolded. (Berg). There are too many possible structures for a random
process (Levinthal’s paradox, 5 x 10
47

structures for 100 aa protein).
Progressive stabilization of intermediates results in correctly folded
proteins.

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Molecular chaperones stabilize hydrophobic sequences of
newly synthesized polypeptides to enable orderly folding


Improperly folded proteins do not exit the ER


HSPs: heat shock proteins, so named because their expression increases in response to heat and other
cellular stresses that result in buildup of mis
-
folded proteins


HSPs require energy.


Polypeptides carry the information to fold in their sequences with assistance from chaperones

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The GroEL/GroES (Hsp60/Hsp10) chaperone machine

Richardson, A., Landry, S. J., and Georgopoulos, C. (1998)
Trends Biochem Sci

23
, 138
-
43.


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Molecular chaperones and protein folding quality control

example: calnexin and protein
N
-
glycosylation

Lodish

The role of
N
-
linked glycosylation in ER
protein folding. The ER
-
membrane
-
bound
chaperone protein calnexin binds to
incompletely folded proteins containing
one terminal glucose on
N
-
linked
oligosaccharides, trapping the protein in
the ER. Removal of the terminal glucose
by a glucosidase releases the protein from
calnexin. A glucosyl transferase is the
crucial enzyme that determines whether
the protein is folded properly or not: if the
protein is still incompletely folded, the
enzyme transfers a new glucose from
UDP
-
glucose to the
N
-
linked
oligosaccharide, renewing the protein's
affinity for calnexin and retaining it in the
ER. The cycle repeats until the protein has
folded completely. Calreticulin functions
similarly, except that it is a soluble ER
resident protein. Another ER chaperone,
ERp57 (not shown), collaborates with
calnexin and calreticulin in retaining an
incompletely folded protein in the ER.

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The protein folding problem: can we predict the three dimensional
structure of a protein from its amino acid sequence


The sequence contains the information necessary for folding


Useful predictions of secondary structure can be made (numerous tools on
web, Expasy)


A given peptide sequence may produce more than one fold in different
proteins


Conformational preferences of AAs not absolute


Tertiary interactions among residues far apart in sequence influence the
formation of secondary structure.


The integration of secondary structures is a very computationally intensive
problem.


There is steady progress in understanding polypeptide properties but no
clear solution to the protein folding problem (Nobel Prize!)


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Free energy change of protein folding:


Unfolded proteins are random, folding
entails considerable increase in order (so,
why does it occur spontaneously?)




Water molecules must form highly ordered
cages around hydrophobic aa residues.
Folding shields these residues from water,
balancing the apparent increase in order.


H
-
bonding, electrostatic and Van der
Waal’s interactions results in a release in
heat (negative enthalpy,
Δ
H)

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Lesk, chap 5 Fig. 8

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Quaternary structure: spatial arrangement of multi subunit
proteins (made of more than one polypeptide chain)

A simple dimer: Quaternary
Structure. The Cro protein of
bacteriophage
λ

is a dimer of
identical subunits.

A hetero
-
tetramer: hemoglobin is composed of 2
α

and 2
β

chains, each with a heme group.

Quaternary structure results from numerous interactions between the
surfaces of the protein molecules. Structural plasticity allows
cooperative oxygen binding in hemoglobin. Molecular machines are
multiprotein complexes that execute many of the important functions
in the cell (ribosome, nuclear pore complex, etc)

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Protein
quaternary
structure

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Atomic structure of the 50S Ribosome Subunit.
Proteins are shown in blue and the two RNA
strands in orange and yellow. The small patch
of green in the center of the subunit is the active
site. (Wikipedia)

Micro
-
environments and macromolecular complexes

The eukaryotic membrane, showing lipid bilayer, integral
membrane proteins, protein channel, glycolipids

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X
-
Ray Crystallography

Steps:

1.
Recombinant expression of protein

2.
Formation of large, pure crystals,
regular in structure, no imperfections

3.
X
-
ray exposure, measurement of
diffraction pattern, as crystal is
rotated

4.
Computation on raw data,
refinement, model building

5.
Repeat



~36,000 protein structures solved to date
using X
-
ray crystallography


Crystal formation difficult for membrane
proteins


Very bright X
-
ray source needed
(synchrotron) to produce the highest
resolution (national labs)

Wikipedia

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NMR Spectroscopy

6000 protein structures solved

Wuthrich, K. (1990)
J Biol Chem

265
, 22059
-
22062.



Nuclear magnetic resonance measures the
environment of protons and other nuclei (
13
C,
15
N,
31
P)


CH, NH, OH, COOH, etc


NMR experiments determine distance
constraints between NMR active nuclei in
biomolecules


High concentration, high purity protein needed


Able to measure protein dynamics


Limited ability to solve structures of very large
proteins


Expression of isotope enriched proteins to
maximize NMR sensitivity


Requires a very large magnet (900 MHz NMR
spectrometer, above)


Resource and computationally intensive