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09/11/08Biochemistry: Protein Function
Protein Function
Andy HowardIntroductory Biochemistry, Fall 2008
11 September 2008
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Topics for today Zymogens and
Post-translational modification
Allostery Specific protein
functions Structural proteins Enzymes Electron transport
Specific functions (continued) Storage & transport
Proteins Hormones &
receptors Nucleic-acid binding
proteins Other functions
Distributions
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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
Bacillus amylo-liquifaciensSubtilisin prosegment complexed with subtilisinPDB 1spb2.0Å29.2+8.6 kDa
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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
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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
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iClicker question 2Which 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.
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iClicker question 3Why 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.
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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
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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
v0
[S]
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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
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Other effectors can influence RT transitions
Post-translational covalent modifiers often influence RT equilibrium Phosphorylation can stabilize either the
R or T state Binding of downstream products can
inhibit TR transition Binding of alternative metabolites can
stabilize R state
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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!
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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!
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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
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Classes of proteins Remainder of this lecture:
small encyclopedia of theprotein functions
Be aware of the fact thatproteins can take onmore than one function A protein may evolve for one purpose … then it gets co-opted for another Moonlighting proteins (Jeffery et al,
Tobeck)
Arginosuccinate lyase /Delta crystallinPDB 1auw, 2.5Å206kDa tetramer
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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)
Examples: collagen, fibroin, keratin Often enzymes are recruited to
perform structural roles
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are needed to see this picture.
CollagenmodelPDB 1K6F
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Enzymes
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
hen egg-white lysozymePDB 2vb10.65Å, 14.2kDa
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are needed to see this picture.
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Many enzymes are oligomeric
Both heterooligomers and homooligomers ADH: tetramer of identical
subunits RuBisCO: 8 identical large
subunits, 8 identical small subunits
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PDB 2hcy: tetramer
PDB 1ej7: 2.45Å8*(13.5+52.2kDa)
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IUBMB Major Enzyme ClassesEC # Class Reactions Sample Comments
1 oxidoreductases Oxidation-reduction
LDH NAD,FMN
2 transferases Transfer big group
AAT Includes kinases
3 hydrolases Transfer of H2O
Pyrophos hydrolase
Includes proteases
4 lyases Addition across =
Pyr decar-boxylase
synthases
5 isomerases Unimolec-ular rxns
Alanineracemase
Includesmutases
6 ligases Joining 2 substrates
Gln synthetase
Often need ATP
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Electron-transport proteins
Involved in Oxidation-reductionreactions via Incorporated metal ions 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
Recombinant human cytochrome cPDB 1J3SNMR structure11.4kDa
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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
AnacystisflavodoxinPDB 1czn1.7Å18.6 kDa
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Storage and transport proteins
Hemoglobin, myoglobin classic examples “honorary enzymes”: share some
characteristics with enzymes 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!
Sperm-whale myoglobin
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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 So storage proteins provide ways
of encapsulating small molecules until they’re needed; then they’re released
T.maritimaferritinPDB 1z4a8*(18 kDa)
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Hormones
Transported signaling molecules,secreted by one tissue and detectedby 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 some are organic, non-peptidic moieties Others: peptide oligomers, too small to be proteins But some hormones are in fact normal-sized proteins.
Human insulinPDB 1t1k3.3+2.3 kDa
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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
of the membrane Membrane part often helical:
usually odd # of spanning helices (7?)
Retinal from bacteriorhodopsinPDB 1r2nNMR structure27.4 kDa
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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!
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Nucleic-acid binding proteins
Many enzymes interact with RNA or DNA But there are non-catalytic proteins that
also bind nucleic acids Scaffolding for ribosomal activity Help form molecular machines for replication,
transcription, RNA processing: These often involve interactions with specific
bases, not just general feel-good interactions Describe these as “recognition steps”
Human hDim1PDB 1pqnNMR struct.14kDa
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Scaffolding(adapter) proteins
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
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Human regulatory complex(Crk SH2 + Abl SH3)PDB 1JU5NMR structure
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Protective proteins
Eukaryotic protective proteins: Immunoglobulins Blood-clotting proteins
(activated by proteolytic cleavage)
Antifreeze proteins
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E5 Fragment of bovine fibrinogenPDB 1JY2, 1.4Å2*(5.3+6.2+5.8) kDa
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Other protective and exploitive proteins Plant, bacterial, and
snake-venom toxins Ricin, abrin (plant
proteins that discourage predation by herbivores)
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Synthetic Abrin-APDB 1ABR2.14Å29.3+27.6 kDa
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Vibrio cholerae toxin A1 + ARF6PDB 2A5F2.1Å21.2+19.3 kDa
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Special functions
Monellin: sweet protein Resilin: ultra-elastic insect wing protein Glue proteins (barnacles, mussels)
Adhesive ability derived from DOPA crosslinks
Potential use in wound closure!
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Dioscoreophyllum MonellinPDB 1KRL5.5+4.8 kDa
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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
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Protein FunctionsFig.15 from Venter et al. (2001), Science 291:1304