Protein Structure concluded;
Protein Function
Andy Howard
Introductory Biochemistry, Fall 2007
19 September 2007
Protein Structure Helps us
Understand Protein Function
If we do know what a protein does,
its structure will tell us how it does it.
 If we don’t know what a protein
does, its structure might give us
what we need to know to figure out
its function.
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IIT Biochemistry: 19 Sep 2007
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Welcome to:
Talk like a Pirate Day
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Undergraduate dorms have
enthusiastically participated
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http://www.talklikeapirate.com/
Also note
http://www.boundingmain.com/
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IIT Biochemistry: 19 Sep 2007
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Plans for Today
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Protein
Structure,
Concluded
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Tertiary Structure
Domains
Protein Function
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Structure-function
relationships
Zymogens
IIT Biochemistry: 19 Sep 2007
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Classes of proteins
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Structural proteins
Enzymes
Electron-transport proteins
Storage and transport
proteins
Hormones
Receptors
Nucleic-acid-binding
proteins
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Secondary structure in
globular proteins
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Segments with secondary structure are usually
short: 2-30 residues
Some globular proteins are almost all helical,
but even then there are bends between short
helices
Other proteins: mostly beta
Others: regular alternation of , 
Still others: irregular , , “coil”
IIT Biochemistry: 19 Sep 2007
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Protein Topology
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Description of the
connectivity of
segments of
secondary structure
and how they do or
don’t cross over
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TIM barrel
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Alternating ,  creates parallel pleated sheet
Bends around as it goes to create
barrel
IIT Biochemistry: 19 Sep 2007
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How do we visualize protein
structures?
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It’s often as important to decide what to
omit as it is to decide what to include
Any segment larger than about 10Å
needs to be simplified if you want to
understand it
What you omit depends on what you
want to emphasize
IIT Biochemistry: 19 Sep 2007
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Styles of protein depiction
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All atoms
All non-H atoms
Main-chain (backbone) only
One dot per residue (typically at C)
Ribbon diagrams:
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Helical ribbon for helix
Flat ribbon for strand
Thin string for coil
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Ribbon diagrams
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Mostly helical:
RecG - DNA
IIT Biochemistry: 19 Sep 2007
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Mixed: lysozyme
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How do we show 3-D?
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Stereo pairs
Dynamics: rotation of flat image
Perspective (hooray, Renaissance)
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Stereo pair: Release factor 2/3
Klaholz et al, Nature (2004) 427:862
IIT Biochemistry: 19 Sep 2007
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A more pedestrian application
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Sso7d bound to DNA
Gao et al (1998) NSB 5: 782
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A little more complex:
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Aligning Cytochrome C5
with Cytochrome C550
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Domains
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Proteins (including singlepolypeptide proteins) often
contain roughly selfcontained domains
Domains often separated by
linkers
Linkers sometimes flexible or
extended or both
Cf. fig. 4.23 in Horton
IIT Biochemistry: 19 Sep 2007
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Protein Function: Generalities
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Proteins do a lot of different things. Why?
Well, they’re coded for by the ribosomal
factories
… But that just backs us up to the
question of why the ribosomal
mechanism codes for proteins and not
something else!
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Proteins are chemically nimble
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The chemistry of proteins is flexible
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Protein side chains can participate in many interesting
reactions
Even main-chain atoms can play roles in certain
circumstances.
Wide range of hydrophobicity available (from
highly water-hating to highly water-loving) within
and around proteins gives them versatility that a
more unambiguously hydrophilic species (like
RNA) or a distinctly hydrophobic species (like a
triglyceride) would not be able to acquire.
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What proteins can do
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Proteins can act as catalysts, transporters,
scaffolds, signals, or fuel in watery or greasy
environments, and can move back and forth
between hydrophilic and hydrophobic
situations.
Furthermore, proteins can operate either in
solution, where their locations are undefined
within a cell, or anchored to a membrane.
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Membrane binding keeps them in place.
Function may occur within membrane or in an
aqueous medium adjacent to the membrane
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Structure-function relationships
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Proteins with known function: structure can tell
is how it does its job
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Example: yeast alcohol dehydrogenase
Catalyzes
ethanol + NAD+  acetaldehyde + NADH + H+
We can say something general about the protein and
the reaction it catalyzes without knowing anything
about its structure
But a structural understanding should help us
elucidate its catalytic mechanism
Protein with unknown function: structure might
tell us what the function is!
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Why this example?
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Structures of ADH from
several eukaryotic and
prokaryotic organisms
already known
Yeast ADH is clearly
important and heavily
studied, but there is no highresolution structure of it!
We got crystals 6 years ago,
but so far I haven’t been able
to determine the structure
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What we know about this enzyme
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Cell contains an enzyme that
interconverts ethanol and acetaldehyde,
using NAD as the oxidizing agent (or
NADH as the reducing agent)
We can call it alcohol dehydrogenase or
acetaldehyde reductase; in this instance
the former name is more common, but
that’s fairly arbitrary (contrast with DHFR)
IIT Biochemistry: 19 Sep 2007
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Size and composition
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Tetramer of identical polypeptides
Total molecular mass = 140 kDa
We can do arithmetic: the individual
polypeptides have a molecular mass of
35 kDa (~330 aa).
Human is a bit bigger: 374 aa per subunit
Based on related structures each subunit
is expected to have an NAD-binding
Rossmann fold over part of its structure
IIT Biochemistry: 19 Sep 2007
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Zymogens and PTM
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Many proteins are
synthesized on the ribosome
in an inactive form, viz. as a
zymogen
The conversions that alter the
ribosomally encoded protein
into its active form is an
instance of post-translational
modification
IIT Biochemistry: 19 Sep 2007
Subtilisin
prosegment
complexed with
subtilisin
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Why PTM?
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This happens for several reasons
Active protein needs to bind cofactors, ions,
carbohydrates, and other species
Active protein might be dangerous at the
ribosome, so it’s created in inactive form and
activated elsewhere
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Proteases (proteins that hydrolyze peptide bonds) are
examples of this phenomenon
… but there are others
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Classes of proteins
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Remainder of this lecture:
small encyclopedia of the
protein functions
Be aware of the fact that
proteins can take on
more than one function
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Arginosuccinate lyase /
Delta crystallin
A protein may evolve for one purpose
… then it gets co-opted for another
Moonlighting proteins (Jeffery et al,
Tobeck)
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Structural proteins
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Perform mechanical or scaffolding
tasks
Not involved in chemistry, unless you
consider this to be a chemical
reaction:
(Person standing upright) 
(Person lying in a puddle on the floor)
Examples: collagen, fibroin, keratin
Often enzymes are recruited to
perform structural roles
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Enzymes
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Enzymes are biological catalysts, i.e.
their job is to reduce the activation
energy barrier between substrates
and products
Tend to be at least 12kDa (why? You
need that much scaffolding)
Usually but not always aqueous
Usually organized with hydrophilic
residues facing outward
IIT Biochemistry: 19 Sep 2007
Hen egg-white
lysozyme
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Many enzymes
are oligomeric
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Both heterooligomers
and homooligomers
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ADH: tetramer of identical
subunits
RuBisCO: 8 identical
large subunits, 8 identical
small subunits
IIT Biochemistry: 19 Sep 2007
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Electron-transport
proteins
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Involved in Oxidation-reduction
reactions via
Recombinant human
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Incorporated metal ions
cytochrome c
Small organic moieties (NAD, FAD)
Generally not enzymes because they’re
ultimately altered by the reactions in
which they participate
But they can be considered to participate
in larger enzyme complexes than can
restore them to their original state
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Sizes and
characteristics
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Some ET proteins are fairly small
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flavodoxin
Cytochrome c
Some flavodoxins
Others are multi-polypeptide complexes
Cofactors or metals may be closely
associated (covalent in cytochromes) or
more loosely bound
IIT Biochemistry: 19 Sep 2007
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Storage and
transport proteins
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Hemoglobin, myoglobin classic examples
“honorary enzymes”: share some
characteristics with enzymes
Sperm-whale
myoglobin
Sizes vary widely
Many transporters operate over much
smaller size-scales than hemoglobin (µm vs.
m):often involved in transport across
membranes
We’ll discuss intracellular transport a lot!
IIT Biochemistry: 19 Sep 2007
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Why do we have
storage proteins?
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Many metabolites are toxic in the
wrong places or at the wrong
times
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Oxygen is nasty
Too much Ca2+ or Fe3+ can be
hazardous
Bacterial
ferritin
So storage proteins provide
ways of encapsulating small
molecules until they’re needed;
then they’re released
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Hormones
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Transported signaling molecules,
secreted by one tissue and detected
insulin
by receptors in another tissue
Signal noted by the receptor will trigger some
kind of response in the second tissue.
They’re involved in cell-cell or tissue-to-tissue
communication.
Not all hormones are proteins
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some are organic, non-peptidic moieties
Others: peptide oligomers, too small to be proteins
But some hormones are in fact normal-sized proteins.
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Receptors
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Many kinds, as distinguished by what
they bind:
Some bind hormones, others
metabolites, others non-hormonal
proteins
Retinal from
Usually membrane-associated:
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bacteriorhodopsin
a soluble piece sticking out
Hydrophobic piece in the membrane
sometimes another piece on the other side
of the membrane
Membrane part often helical:
usually odd # of spanning helices (7?)
IIT Biochemistry: 19 Sep 2007
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Why should it work this way?
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Two aqueous
domains, one near N
terminus and the
other near the C
terminus, are
separated by an odd
number of helices
This puts them on
opposite sides of the
membrane!
IIT Biochemistry: 19 Sep 2007
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Nucleic-acid
binding proteins
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Many enzymes interact with RNA or DNA
But there are non-catalytic proteins that
also bind nucleic acids
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Scaffolding for ribosomal activity
Help form molecular machines for replication,
transcription, RNA processing:
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These often involve interactions with specific
bases, not just general feel-good interactions
Describe these as “recognition steps”
IIT Biochemistry: 19 Sep 2007
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DIM1