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Protein Structure &
Function
Andy Howard
Introductory Biochemistry, Fall 2010
7 September 2010
Biochem: Protein Functions I
09/07/2010
Proteins and enzymes
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Proteins perform a variety of
functions, including acting as
enzymes.
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Biochem: Protein Functions I
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Plans for Today
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Secondary Structure
Types
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Helices
Sheets
Disulfides
Tertiary Structures
Quaternary
Structure
Visualizing structure
09/07/2010
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The Protein Data Bank
Tertiary & quaternary
structure
Protein Functions
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Structure-function
relationships
Post-translational
modification
Biochem: Protein Functions I
p. 3 of 52
Components of secondary
structure
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, 310,  helices
pleated sheets
and the strands
that comprise them
Beta turns
More specialized
structures like
collagen helices
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Biochem: Protein Functions I
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An accounting for secondary
structure: phospholipase A2
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Biochem: Protein Functions I
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Alpha helix
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Biochem: Protein Functions I
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Characteristics of  helices
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Hydrogen bonding from amino nitrogen
to carbonyl oxygen in the residue 4
earlier in the chain
3.6 residues per turn
Amino acid side chains face outward, for
the most part
~ 10 residues long in globular proteins
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Biochem: Protein Functions I
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What would disrupt this?
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Not much: the side chains
don’t bump into one another
Proline residue will disrupt
it:
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Main-chain N can’t H-bond
The ring forces a kink
Glycines sometimes disrupt
because they tend to be
flexible
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Biochem: Protein Functions I
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Other helices

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NH to C=O four residues earlier is
not the only pattern found in
proteins
310 helix is NH to C=O three
residues earlier

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More kinked; 3 residues per turn
Often one H-bond of this kind at Nterminal end of an otherwise -helix
  helix: even rarer: NH to C=O
five residues earlier
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Biochem: Protein Functions I
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Beta strands
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Structures containing roughly extended
polypeptide strands
Extended conformation stabilized by interstrand main-chain hydrogen bonds
No defined interval in sequence number
between amino acids involved in H-bond
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Biochem: Protein Functions I
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Sheets: roughly
planar
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Folds straighten H-bonds
Side-chains roughly
perpendicular from sheet
plane
Consecutive side chains
up, then down
Minimizes intra-chain
collisions between bulky
side chains
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Biochem: Protein Functions I
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Anti-parallel
beta sheet
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Neighboring strands extend in opposite
directions
Complementary C=O…N bonds from
top to bottom and bottom to top strand
Slightly pleated for optimal H-bond
strength
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Biochem: Protein Functions I
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Parallel
Beta Sheet
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N-to-C directions are the same for both
strands
You need to get from the C-end of one
strand to the N-end of the other strand
somehow
H-bonds at more of an angle relative to the
approximate strand directions
Therefore: more pleated than anti-parallel
sheet
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Biochem: Protein Functions I
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Beta turns
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Abrupt change in direction
, angles are
characteristic of beta
Main-chain H-bonds
maintained almost all the
way through the turn
Jane Richardson and others
have characterized several
types
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Biochem: Protein Functions I
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Collagen triple helix
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Three left-handed helical
strands interwoven with a
specific hydrogen-bonding
interaction
Every 3rd residue
approaches other strands
closely: so they’re glycines
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Biochem: Protein Functions I
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Note about disulfides
H
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Cysteine residues brought
S
into proximity under
H
C
oxidizing conditions can
form a disulfide
Forms a “cystine” residue
Oxygen isn’t always the
oxidizing agent
Can bring sequence-distant
residues close together and
stabilize the protein
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Biochem: Protein Functions I
H
S
H
H
+
(1/2)O 2
H2O
H
H
C
C
S
H
S
H
p. 16 of 52
H
C
Hydrogen bonds, revisited
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Protein settings, H-bonds are almost always:
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Between carbonyl oxygen and hydroxyl:
(C=O ••• H-O-)
between carbonyl oxygen and amine:
(C=O ••• H-N-)
–OH to –OH, –OH to –NH, … less significant
These are stabilizing structures
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Any stabilization is (on its own) entropically
disfavored;
Sufficient enthalpic optimization overcomes that!
In general the optimization is ~ 1- 4 kcal/mol
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Biochem: Protein Functions I
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Secondary structures in
structural proteins
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Structural proteins often have uniform
secondary structures
Seeing instances of secondary structure
provides a path toward understanding them
in globular proteins
Examples:
Alpha-keratin (hair, wool, nails, …):
-helical
Silk fibroin (guess) is -sheet
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Biochem: Protein Functions I
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Alpha-keratin
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Actual -keratins
sometimes contain helical
globular domains
surrounding a fibrous
domain
Fibrous domain: long
segments of regular helical bonding patterns
Side chains stick out from
the axis of the helix
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Biochem: Protein Functions I
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Silk
fibroin
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Antiparallel
beta sheets
running parallel
to the silk fiber
axis
Multiple repeats
of (Gly-Ser-GlyAla-Gly-Ala)n
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Biochem: Protein Functions I
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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”
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Tertiary Structure
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The overall 3-D arrangement of atoms
in a single polypeptide chain
Made up of secondary-structure
elements & locally unstructured strands
Described in terms of sequence,
topology, overall fold, domains
Stabilized by van der Waals
interactions, hydrogen bonds,
disulfides, . . .
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Biochem: Protein Functions I
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Quaternary
structure
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Arrangement of individual polypeptide
chains to form a complete oligomeric,
functional protein
Individual chains can be identical or
different
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If they’re the same, they can be coded for
by the same gene
If they’re different, you need more than
one gene
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Biochem: Protein Functions I
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Not all proteins have all four
levels of structure
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Monomeric proteins don’t have
quaternary structure
Tertiary structure: subsumed into
2ndry structure for many structural
proteins (keratin, silk fibroin, …)
Some proteins (usually small ones)
have no definite secondary or tertiary
structure; they flop around!
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Biochem: Protein Functions I
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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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Biochem: Protein Functions I
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TIM barrel
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Alternating ,  creates parallel -pleated
sheet
Bends around as it goes to create barrel
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Biochem: Protein Functions I
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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
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Biochem: Protein Functions I
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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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Biochem: Protein Functions I
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How do we show 3-D?
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Stereo pairs
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Rely on the way the brain processes leftand right-eye images
If we allow our eyes to go slightly walleyed or crossed, the image appears
three-dimensional
Dynamics: rotation of flat image
Perspective (hooray, Renaissance)
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Biochem: Protein Functions I
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Straightforward example
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Sso7d bound to DNA
Gao et al (1998) NSB 5: 782
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Biochem: Protein Functions I
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A little more complex:

Aligning Cytochrome C5
with Cytochrome C550
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Biochem: Protein Functions I
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Stereo pair: Release factor 2/3
Klaholz et al, Nature (2004) 427:862
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Biochem: Protein Functions I
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Ribbon diagrams
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Mostly helical:
E.coli RecG - DNA
PDB 1gm5
3.24Å, 105 kDa
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
09/07/2010
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Mixed:
hen egg-white lysozyme
PDB 2vb1
0.65Å, 14.2kDa
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Biochem: Protein Functions I
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The Protein Data Bank
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http://www.rcsb.org/
This is an electronic repository for threedimensional structural information of
polypeptides and polynucleotides
67656 structures as of September 2010
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Most are determined by X-ray crystallography
Smaller number are high-field NMR structures
A few calculated structures, most of which are
either close relatives of experimental structures
or else they’re small, all-alpha-helical proteins
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Biochem: Protein Functions I
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What you can do with
the PDB
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Display structures
Look up specific coordinates
Run clever software that compares
and synthesizes the knowledge
contained there
Use it as a source for determining
additional structures
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Biochem: Protein Functions I
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Generalizations about
Tertiary Structure
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Most globular proteins contain substantial
quantities of secondary structure
The non-secondary segments are usually
short; few knots or twists
Most proteins fold into low-energy
structures—either the lowest or at least in a
significant local minimum of energy
Generally the solvent-accessible surface area
of a correctly folded protein is small
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Biochem: Protein Functions I
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Hydrophobic in, -philic out
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Aqueous proteins arrange themselves so
that polar groups are solvent-accessible
and apolar groups are not
The energetics of protein folding are
strongly driven by this hydrophobic in,
hydrophilic out effect
Exceptions are membrane proteins
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Biochem: Protein Functions I
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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. 6.36 in G&G
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Biochem: Protein Functions I
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Generalizations about
quaternary structure
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Considerable symmetry in many quaternary
structure patterns
(see G&G section 6.5)
Weak polar and solvent-exclusion forces add
up to provide driving force for association
Many quaternary structures are necessary to
function:
often the monomer can’t do it on its own
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Biochem: Protein Functions I
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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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Biochem: Protein Functions I
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Proteins are
chemically nimble
The chemistry of proteins is flexible
 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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Biochem: Protein Functions I
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Structure-function relationships
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Proteins with known function: structure can tell
is how it does its job
 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
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Biochem: Protein Functions I
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Why this example?
Structures of ADH from
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
several eukaryotic and
prokaryotic organisms
already known
Yeast ADH
 Yeast ADH is clearly
PDB 2hcy
important and heavily
2.44Å
152 kDa tetramer
studied, but until 2006: no
dimer shown
structure!
 We got crystals 11 years ago,
but so far I haven’t been able
09/07/2010
p. 43 of 52
Biochem:
Functions I
to determine
theProtein
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)
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Biochem: Protein Functions I
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Size and composition
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Tetramer of identical polypeptides
Total molecular mass = 152 kDa
We can do arithmetic: the individual
polypeptides have a molecular mass of
38 kDa (347 aa).
Human is a bit bigger: 374 aa per subunit
Each subunit has an NAD-binding
Rossmann fold over part of its structure
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Biochem: Protein Functions I
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Structure-function
relationships II
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Protein with unknown function:
structure might tell us what the function
is!
Generally we accomplish this by
recognizing structural similarity to
another protein whose function is
known
Sometimes we get lucky: we can figure
it out by binding of a characteristic
cofactor
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Biochem: Protein Functions I
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What proteins can do: I

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.
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What proteins can do: II
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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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Biochem: Protein Functions I
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What proteins can do: III
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Proteins can readily bind organic, metallic,
or organometallic ligands called cofactors.
These extend the functionality of proteins
well beyond the chemical nimbleness that
polypeptides by themselves can
accomplish
We’ll study these cofactors in detail in
chapter 17
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Biochem: Protein Functions I
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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
09/07/2010
Biochem: Protein Functions I
QuickTime™ and a
decompressor
are needed to see this picture.
PDB 3CNQ
Subtilisin
prosegment
complexed with
subtilisin
1.71Å; 35 kDa
monomer
p. 50 of 52
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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Biochem: Protein Functions I
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Protein Phosphorylation
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Most common form of PTM that affects just
one amino acid at a time
Generally involves phosphorylating side
chains of specific polar amino acids:
mostly S,T,Y,H (and D, E)
Enzymes that phosphorylate proteins are
protein kinases and are ATP or GTP
dependent
Enzymes that remove phosphates are
phosphatases and are ATP and GTP
independent
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