Protein Structure concluded; Protein Function Andy Howard Introductory Biochemistry, Fall 2007 19 September 2007

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.


Welcome to:Talk like a Pirate Day

Undergraduate dorms have enthusiastically participated

http://www.talklikeapirate.com/

Also note http://www.boundingmain.com/


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


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”


Protein Topology Description of the connectivity of segments of secondary structure and how they do or don’t cross over


TIM barrel Alternating , creates parallel -pleated sheet

Bends around as it goes to create barrel


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


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


Ribbon diagrams Mostly helical:RecG - DNA

Mixed: lysozyme


How do we show 3-D?

Stereo pairs Dynamics: rotation of flat image

Perspective (hooray, Renaissance)


Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862


A more pedestrian application

Sso7d bound to DNAGao et al (1998) NSB 5: 782


A little more complex: Aligning Cytochrome C5with Cytochrome C550


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


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!


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.


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


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!


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


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)


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


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


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


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


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


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


Many enzymes are oligomeric

Both heterooligomers and homooligomers ADH: tetramer of identical subunits

RuBisCO: 8 identical large subunits, 8 identical small subunits


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


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


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


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


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


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


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!


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

Documents

Protein Structure concluded; Protein Function Andy Howard Introductory Biochemistry, Fall 2007 19 September 2007