Protein Structure & Function

09/07/2010Biochem: Protein Functions I

Protein Structure & Function

Andy HowardIntroductory Biochemistry,

Fall 2010 7 September 2010

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Biochem: Protein Functions I p. 2 of 52

Proteins and enzymes

Proteins perform a variety of functions, including acting as enzymes.

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Plans for Today

Secondary Structure Types Helices Sheets

Disulfides Tertiary Structures

Quaternary Structure

Visualizing structure

The Protein Data Bank

Tertiary & quaternary structure

Protein Functions Structure-function relationships

Post-translational modification

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Components of secondary structure , 310, helices pleated sheets and the strands that comprise them

Beta turns More specialized structures like collagen helices

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An accounting for secondary structure: phospholipase A2

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Alpha helix

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Characteristics of helices

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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What would disrupt this?

Not much: the side chains don’t bump into one another

Proline residue will disrupt it: 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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Other helices 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 More kinked; 3 residues per turn

Often one H-bond of this kind at N-terminal end of an otherwise -helix

helix: even rarer: NH to C=O five residues earlier

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Beta strands

Structures containing roughly extended polypeptide strands

Extended conformation stabilized by inter-strand main-chain hydrogen bonds

No defined interval in sequence number between amino acids involved in H-bond

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Sheets: roughly planar

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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Anti-parallel beta sheet 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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Parallel Beta Sheet

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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Beta turns Abrupt change in direction

, angles arecharacteristic 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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Collagen triple helix

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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Note about disulfides

Cysteine residues brought into proximity under 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

CHHSHCHHSH+(1/2)O2SSHCHHCHH2O

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Hydrogen bonds, revisited

Protein settings, H-bonds are almost always: 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

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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Secondary structures in structural proteins

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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Alpha-keratin 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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Silk fibroin Antiparallel

beta sheets running parallel to the silk fiber axis

Multiple repeats of (Gly-Ser-Gly-Ala-Gly-Ala)n

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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”

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Tertiary Structure

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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Quaternary structure

Arrangement of individual polypeptide chains to form a complete oligomeric, functional protein

Individual chains can be identical or different 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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Not all proteins have all four levels of structure 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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Protein Topology

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

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TIM barrel Alternating , creates parallel -pleated sheet

Bends around as it goes to create barrel

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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

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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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How do we show 3-D? Stereo pairs

Rely on the way the brain processes left- and right-eye images

If we allow our eyes to go slightly wall-eyed or crossed, the image appears three-dimensional

Dynamics: rotation of flat image Perspective (hooray, Renaissance)

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Straightforward example Sso7d bound to DNA

Gao et al (1998) NSB 5: 782

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A little more complex: Aligning Cytochrome C5with Cytochrome C550

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

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Ribbon diagrams Mostly helical:

E.coli RecG - DNA PDB 1gm5

3.24Å, 105 kDa

Mixed:hen egg-white lysozyme

PDB 2vb10.65Å, 14.2kDa

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.



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The Protein Data Bank

http://www.rcsb.org/ This is an electronic repository for three-dimensional structural information of polypeptides and polynucleotides

67656 structures as of September 2010 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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What you can do with the PDB 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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Generalizations about Tertiary Structure 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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Hydrophobic in, -philic out 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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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. 6.36 in G&G

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Generalizations about quaternary structure 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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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!

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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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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

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Why this example?

Structures of ADH from several eukaryotic and prokaryotic organisms already known

Yeast ADH is clearly important and heavily studied, but until 2006: no structure!

We got crystals 11 years ago, but so far I haven’t been able to determine the structure



Yeast ADHPDB 2hcy2.44Å152 kDa tetramerdimer shown

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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)

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Size and composition 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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Structure-functionrelationships II 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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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 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

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What proteins can do: III

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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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

PDB 3CNQSubtilisin prosegment complexed with subtilisin1.71Å; 35 kDa monomer

QuickTime™ and a decompressor


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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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Protein Phosphorylation 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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Protein Structure & Function