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Protein Structure concluded;
Protein Function
Andy HowardIntroductory Biochemistry,
Fall 200719 September 2007
IIT Biochemistry: 19 Sep 2007 Slide 2 of 36
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.
IIT Biochemistry: 19 Sep 2007 Slide 3 of 36
Welcome to:Talk like a Pirate Day
Undergraduate dorms have enthusiastically participated
http://www.talklikeapirate.com/
Also note http://www.boundingmain.com/
IIT Biochemistry: 19 Sep 2007 Slide 4 of 36
Plans for Today
Protein Structure, Concluded Tertiary Structure
Domains Protein Function
Structure-function relationships
Zymogens
Classes of proteins Structural proteins Enzymes Electron-transport proteins
Storage and transport proteins
Hormones Receptors Nucleic-acid-binding proteins
IIT Biochemistry: 19 Sep 2007 Slide 5 of 36
Secondary structure in globular proteins 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 Slide 6 of 36
Protein Topology Description of the connectivity of segments of secondary structure and how they do or don’t cross over
IIT Biochemistry: 19 Sep 2007 Slide 7 of 36
TIM barrel Alternating , creates parallel -pleated sheet
Bends around as it goes to create barrel
IIT Biochemistry: 19 Sep 2007 Slide 8 of 36
How do we visualize protein structures? 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 Slide 9 of 36
Styles of protein depiction
All atoms All non-H atoms Main-chain (backbone) only One dot per residue (typically at C)
Ribbon diagrams: Helical ribbon for helix Flat ribbon for strand Thin string for coil
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Ribbon diagrams Mostly helical:RecG - DNA
Mixed: lysozyme
IIT Biochemistry: 19 Sep 2007 Slide 11 of 36
How do we show 3-D?
Stereo pairs Dynamics: rotation of flat image
Perspective (hooray, Renaissance)
IIT Biochemistry: 19 Sep 2007 Slide 12 of 36
Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862
IIT Biochemistry: 19 Sep 2007 Slide 13 of 36
A more pedestrian application
Sso7d bound to DNAGao et al (1998) NSB 5: 782
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A little more complex: Aligning Cytochrome C5with Cytochrome C550
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Domains
Proteins (including single-polypeptide proteins) often contain roughly self-contained domains
Domains often separated by linkers
Linkers sometimes flexible or extended or both
Cf. fig. 4.23 in Horton
IIT Biochemistry: 19 Sep 2007 Slide 16 of 36
Protein Function: Generalities 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!
IIT Biochemistry: 19 Sep 2007 Slide 17 of 36
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.
IIT Biochemistry: 19 Sep 2007 Slide 18 of 36
What proteins can do 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. Membrane binding keeps them in place. Function may occur within membrane or in an aqueous medium adjacent to the membrane
IIT Biochemistry: 19 Sep 2007 Slide 19 of 36
Structure-function relationships Proteins with known function: structure can tell is how it does its job Example: yeast alcohol dehydrogenase Catalyzesethanol + 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!
IIT Biochemistry: 19 Sep 2007 Slide 20 of 36
Why this example? Structures of ADH from several eukaryotic and prokaryotic organisms already known
Yeast ADH is clearly important and heavily studied, but there is no high-resolution structure of it!
We got crystals 6 years ago, but so far I haven’t been able to determine the structure
IIT Biochemistry: 19 Sep 2007 Slide 21 of 36
What we know about this enzyme 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 Slide 22 of 36
Size and composition 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 Slide 23 of 36
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
Subtilisin prosegment complexed with subtilisin
IIT Biochemistry: 19 Sep 2007 Slide 24 of 36
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
IIT Biochemistry: 19 Sep 2007 Slide 25 of 36
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 crystallin
IIT Biochemistry: 19 Sep 2007 Slide 26 of 36
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
IIT Biochemistry: 19 Sep 2007 Slide 27 of 36
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 lysozyme
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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
IIT Biochemistry: 19 Sep 2007 Slide 29 of 36
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 c
IIT Biochemistry: 19 Sep 2007 Slide 30 of 36
Sizes and characteristics Some ET proteins are 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
flavodoxin
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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
IIT Biochemistry: 19 Sep 2007 Slide 32 of 36
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
Bacterial ferritin
IIT Biochemistry: 19 Sep 2007 Slide 33 of 36
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.
insulin
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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 bacteriorhodopsin
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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!
IIT Biochemistry: 19 Sep 2007 Slide 36 of 36
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”
DIM1