Protein Structure

advertisement
Protein Function
Andy Howard
Introductory Biochemistry, Fall 2008
11 September 2008
Biochemistry: Protein Function
09/11/08
Topics for today



Zymogens and
Post-translational
modification
Allostery
Specific protein
functions



Structural proteins
Enzymes
Electron transport
Biochemistry: Protein Function

Specific functions
(continued)





Storage & transport
Proteins
Hormones &
receptors
Nucleic-acid binding
proteins
Other functions
Distributions
09/11/08
Page 2 of 33
Zymogens and PTM


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
Biochemistry: Protein Function
09/11/08
Bacillus amyloliquifaciens
Subtilisin
prosegment
complexed
with subtilisin
PDB 1spb
2.0Å
29.2+8.6 kDa
Page 3 of 33
Why PTM?



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


Proteases (proteins that hydrolyze peptide bonds)
are examples of this phenomenon
… but there are others
Biochemistry: Protein Function
09/11/08
Page 4 of 33
iClicker question 1
Why are digestive proteases usually synthesized
as inactive zymogens?
 (a) Because they are produced in one organ
and used elsewhere
 (b) Because that allows the active form to be
smaller than the ribosomally encoded form
 (c) To allow for gene amplification and diversity
 (d) So that the protease doesn’t digest itself
prior to performing its intended digestive
function
 (e) None of the above
Biochemistry: Protein Function
09/11/08
Page 5 of 33
iClicker question 2
Which amino acids can be readily
phosphorylated by kinases?
 (a) asp, phe, gly, leu
 (b) ser, thr, tyr, his
 (c) leu, ile, val, phe
 (d) arg, lys, gln, asn
 (e) none of the above.
Biochemistry: Protein Function
09/11/08
Page 6 of 33
iClicker question 3
Why are kinase reactions ATP- (or GTP-)
dependent, whereas phosphorylase reactions
are not?
 (a) To ensure stereospecific addition of
phosphate to the target
 (b) To prevent wasteful hydrolysis of product
 (c) Adding phosphate is endergonic; taking
phosphate off is exergonic
 (d) None of the above.
Biochemistry: Protein Function
09/11/08
Page 7 of 33
Allostery



Formal definition:
alterations in protein function that occur
when the structure changes upon binding
of small molecules
In practice: often the allosteric effector is
the same species as the substrate:
they’re homotropic effectors
… but not always: allostery becomes an
effective way of characterizing third-party
(heterotropic) activators and inhibitors
Biochemistry: Protein Function
09/11/08
Page 8 of 33
v0
[S]
What allostery means


Non-enzymatic proteins can be
allosteric:
hemoglobin’s affinity for O2 is
influenced by the binding of O2 to
other subunits
In enzymes: non-Michaelis-Menten
kinetics (often sigmoidal) when the
allosteric activator is also the
substrate
Biochemistry: Protein Function
09/11/08
Page 9 of 33
R and T states




Protein with multiple substrate binding sites is
in T (“tense”) state in absence of ligand or
substrate
Binding of ligand or substrate moves enzyme
into R (“relaxed”) state where its affinity for
substrate at other sites is higher
Binding affinity or enzymatic velocity can then
rise rapidly as function of [S]
Once all the protein is converted to R state,
ordinary hyperbolic kinetics take over
Biochemistry: Protein Function
09/11/08
Page 10 of 33
Other effectors can influence
RT transitions

Post-translational covalent modifiers
often influence RT equilibrium



Phosphorylation can stabilize either the
R or T state
Binding of downstream products can
inhibit TR transition
Binding of alternative metabolites can
stabilize R state
Biochemistry: Protein Function
09/11/08
Page 11 of 33
Why does that make sense?


Suppose reactions are:
(E)
ABCD
Binding D to enzyme E (the enzyme that
converts A to B) will destabilize its R
state, limiting conversion of A to B and
(ultimately) reducing / stabilizing [D]:
homeostasis!
Biochemistry: Protein Function
09/11/08
Page 12 of 33
Alternative pathways
• Often one metabolite has two possible
fates:
BCD
A
HIJ
• If we have a lot of J around, it will bind to
the enzyme that converts A to B and
activate it; that will balance D with J!
Biochemistry: Protein Function
09/11/08
Page 13 of 33
How does this work
structurally?


In general, binding of the allosteric
effector causes a medium-sized (~2-5Å)
shift in the conformation of the protein
This in turn alters its properties




Affinity for the ligand
Flexibility (R vs T)
Other properties
We’ll revisit this when we do enzymology
Biochemistry: Protein Function
09/11/08
Page 14 of 33
Classes of proteins


Remainder of this lecture:
small encyclopedia of the
protein functions
Be aware of the fact that
proteins can take on
more than one function



Arginosuccinate lyase /
Delta crystallin
PDB 1auw, 2.5Å
206kDa tetramer
A protein may evolve for one purpose
… then it gets co-opted for another
Moonlighting proteins (Jeffery et al,
Tobeck)
Biochemistry: Protein Function
09/11/08
Page 15 of 33
Structural proteins




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)
QuickTime™ and a
Examples: collagen, fibroin, keratin TIFF (Uncompressed)
decompressor
are needed to see this picture.
Often enzymes are recruited to
Collagen
perform structural roles
model
PDB 1K6F
Biochemistry: Protein Function
09/11/08
Page 16 of 33
Enzymes




QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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
Biochemistry: Protein Function
09/11/08
hen egg-white
lysozyme
PDB 2vb1
0.65Å, 14.2kDa
Page 17 of 33
Many enzymes
are oligomeric

Both heterooligomers
and homooligomers


QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
PDB 2hcy: tetramer
ADH: tetramer of identical
subunits
RuBisCO: 8 identical
large subunits, 8 identical
small subunits
PDB 1ej7: 2.45Å
8*(13.5+52.2kDa)
Biochemistry: Protein Function
09/11/08
Page 18 of 33
IUBMB Major Enzyme Classes
EC # Class
Reactions
Sample
Comments
1
oxidoreductases
Oxidationreduction
LDH
NAD,FMN
2
transferases
Transfer
big group
AAT
Includes
kinases
3
hydrolases
Transfer of
H 2O
Pyrophos
hydrolase
Includes
proteases
4
lyases
Addition
across =
Pyr decarboxylase
synthases
5
isomerases
Unimolecular rxns
Alanine
racemase
Includes
mutases
6
ligases
Joining 2
substrates
Gln
synthetase
Often need
ATP
Biochemistry: Protein Function
09/11/08
Page 19 of 33
Electron-transport
proteins

Involved in Oxidation-reduction
reactions via

Incorporated metal ions
Small organic moieties (NAD, FAD)
Recombinant
human

cytochrome c
 Generally not enzymes because they’re
PDB 1J3S
ultimately altered by the reactions in
NMR structure
which they participate
11.4kDa

But they can be considered to participate
in larger enzyme complexes than can
restore them to their original state
Biochemistry: Protein Function
09/11/08
Page 20 of 33
Sizes and
characteristics

Some ET proteins: fairly small




Cytochrome c
Some flavodoxins
Others are multi-polypeptide
complexes
Cofactors or metals may be
closely associated (covalent in
cytochromes) or more loosely
bound
Biochemistry: Protein Function
09/11/08
Anacystis
flavodoxin
PDB 1czn
1.7Å
18.6 kDa
Page 21 of 33
Storage and
transport proteins





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!
Biochemistry: Protein Function
09/11/08
Page 22 of 33
Why do we have
storage proteins?

Many metabolites are toxic in the
wrong places or at the wrong
times



Oxygen is nasty
Too much Ca2+ or Fe3+ can be
hazardous
T.maritima
ferritin
PDB 1z4a
8*(18 kDa)
So storage proteins provide
ways of encapsulating small
molecules until they’re needed;
then they’re released
Biochemistry: Protein Function
09/11/08
Page 23 of 33
Hormones




Transported signaling molecules,
secreted by one tissue and detected
Human
by receptors in another tissue
insulin
Signal noted by the receptor will trigger some PDB 1t1k
kind of response in the second tissue.
3.3+2.3 kDa
They’re involved in cell-cell or tissue-to-tissue
communication.
Not all hormones are proteins



some are organic, non-peptidic moieties
Others: peptide oligomers, too small to be proteins
But some hormones are in fact normal-sized proteins.
Biochemistry: Protein Function
09/11/08
Page 24 of 33
Receptors



Many kinds, as distinguished by what
they bind:
Some bind hormones, others
metabolites, others non-hormonal
proteins
Usually membrane-associated:
a soluble piece sticking out
 Hydrophobic piece in the membrane
 sometimes another piece on the other side Retinal from
of the membrane
bacteriorhodopsin
PDB 1r2n
 Membrane part often helical:
usually odd # of spanning helices (7?) NMR structure
27.4 kDa

Biochemistry: Protein Function
09/11/08
Page 25 of 33
Why should it work this way?


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!
Biochemistry: Protein Function
09/11/08
Page 26 of 33
Nucleic-acid
binding proteins


Many enzymes interact with RNA or DNA
But there are non-catalytic proteins that
Human hDim1
also bind nucleic acids
PDB 1pqn


Scaffolding for ribosomal activity
Help form molecular machines for replication,
transcription, RNA processing:


NMR struct.
14kDa
These often involve interactions with specific
bases, not just general feel-good interactions
Describe these as “recognition steps”
Biochemistry: Protein Function
09/11/08
Page 27 of 33
Scaffolding
(adapter) proteins



QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Human
regulatory
complex
(Crk SH2 + Abl
SH3)
PDB 1JU5
NMR structure
A type of signaling protein
(like hormones and receptors)
Specific modules of the protein
recognize and bind other proteins:
protein-protein interactions
They thereby function as scaffolds
on which a set of other proteins can
attach and work together
Biochemistry: Protein Function
09/11/08
Page 28 of 33
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Protective proteins

Eukaryotic protective proteins:



Immunoglobulins
Blood-clotting proteins
(activated by proteolytic
cleavage)
Antifreeze proteins
Biochemistry: Protein Function
09/11/08
E5 Fragment of
bovine fibrinogen
PDB 1JY2, 1.4Å
2*(5.3+6.2+5.8)
kDa
Page 29 of 33
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Other protective and
exploitive proteins Vibrio
cholerae toxin


Plant, bacterial, and A1 + ARF6
PDB 2A5F
snake-venom toxins 2.1Å
21.2+19.3
Ricin, abrin (plant
kDa
proteins that discourage
predation by herbivores)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Synthetic Abrin-A
PDB 1ABR
2.14Å
29.3+27.6 kDa
Biochemistry: Protein Function
09/11/08
Page 30 of 33
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Special functions



Dioscoreophyllum
Monellin
PDB 1KRL
5.5+4.8 kDa
Monellin: sweet protein
Resilin: ultra-elastic insect wing protein
Glue proteins (barnacles, mussels)


Adhesive ability derived from DOPA
crosslinks
Potential use in wound closure!
Biochemistry: Protein Function
09/11/08
Page 31 of 33
What percentages do what?





See fig. 5.32 in G&G
42% of all human proteins have unknown
function!
Enzymes are about 20% of proteins with known
functions (incl. 3% kinases, 7.5% nucleic acid
enzymes)
Structural proteins 4.2%
Percentages here reflect diversity, not mass
Biochemistry: Protein Function
09/11/08
Page 32 of 33
Protein Functions
Biochemistry: Protein Function
Fig.15 from Venter et
al. (2001), Science
291:1304
09/11/08
Page 33 of 33
Download