The Three-Dimensional Structure of Proteins - Chapter 4

Protein Classification by SizeProteins: polymers of amino acids•

Contains 10 or less amino acids.-

Molecular weight 1100-

Simplest classification -

Peptide (small)•

Has a molecular weight less than 10 kDa.-

Molecular weight 10,000-

About 90 amino acids-

Polypeptide (medium)•

Has a molecular weigh greater than 10 kDa.-

Molecular weight 10,000 - 500,000+○

Molecular weight 10,000-

90 amino acids-

Protein (large)•

kDa based on gel electrophoresis •Think about molecular weight because the exact cut off of the number of amino acids gets blurry •

Protein StructureOrganized by Increasing Levels of Structural Complexity. •

Protein Primary Structure (1° Structure)

The linear sequence of amino acids in a protein.•Amino acids are listed from the amino terminus to the carboxyl terminus.•Simplest level = primary•Peptide, polypeptide, protein has primary structure•Lys = N-T and Ala = C.T•

Peptide Bonds Link Amino acids in a ProteinHow (why) higher levels of structures form

No free rotation about double bonds (amide link)•

In the end the peptide bond resembles the middle picture -

Amide linkage is subject to resonance stabilization •

Yvonne LeBiochem I Test 2

2011

The Three-Dimensional Structure of Proteins - Chapter 4Wednesday, October 12, 2011

Biochem 1 - Test 2

Amino acids linked together by amide bond = peptide bond•

N has a slightly positive charge and O has slightly negative charge ○

Polar-

Planar because of sp2 hybridization ○

Trans configuration○

Because carbon is in partial double bond ○

Planar-

Since it has partial double bond character, stereochemistry resembles double bonds-

Amide bond with partial double-bond character:•

Trans = substituent groups on opposite sides of the double bond-

Cis = substituent groups on same side of the double bond -

All peptide bonds in proteins will be in trans configuration because it is the most stable ( lowest energy)•

All true (will be in trans conformation all the time) except for proline•

Because proline has small volume-

Unique conformation that allows unique conformation (because alpha carbon is in cyclic configuration)-

Proline: trans and cis = equally stable•

Switching mechanism -

Proline can transfer between cis and trans on conformational change in a protein•

You will mostly see it as trans in a peptide bond •

FIGURE 4-2b The planar peptide group. (b) Three bonds separate sequential α carbons in a polypeptide chain. The N—Cα and Cα—C bonds can rotate, described by dihedral angles designated Φ and Ψ, respectively. The peptide C—N bond is not free to rotate. Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups.

No free rotation about peptide bond but there is rotation about 2 other bonds•

FIGURE 4-7b Structures of β turns. (b) Trans and cis isomers of a peptide bond involving the imino nitrogen of proline. Of the peptide bonds between amino acid residues other than Pro, more than 99.95% are in the trans configuration. For peptide bonds involving the imino nitrogen of proline, however, about 6% are in the cis configuration; many of these occur at β turns.

Biochem 1 - Test 2

ɸ - angle of rotation between Cα and Nα-

Ψ - angle of rotation between Cα and C=Oα-

No free rotation about peptide bond but there is rotation about 2 other bonds•

Results in dihedral angels -

This repositions the R groups in space-

No free rotation in amide bonds but planes of amide bonds can rotate past each other•

Peptide will seek out lowest energy 3D conformation it can find by rotation about ɸ and Ψ-

Peptide in solution : rotation about ɸ and Ψ•

Rotation about Φ and Ψ Leads to High- and Low-Energy Conformations

When rotating R groups can interact with one another •

Overlap of van der waals radii = repulsive (electron clouds overlap and repulse increases energy)○

As rotation occurs R groups can interact (attractively or repulsively)○

Why does rotation lead to high and low energy conformations?-

Rotation will continue until the peptide gets low energy conformation ○

Rotation gives different energy structures - each configuration has a different free energy based in interactions within the peptide -

Rotation about ɸ and Ψ gives different 3D structure - different energies•

Guess work○

They built model peptides : tried to predict what would be low energy 3D conformations -

Alpha helix1.Beta sheet2.Random coil 3.

Pauling and Corey - 20th century•

Ramachandran Plot of Rotation vs. Energy Yields Stable Conformations

Biochem 1 - Test 2

The darker the blue, the lower the energy•

Holes on a punch card-

Computer : took model peptides and entered in rotations about ɸ and Ψ and the computer rotated the model peptide and calculated the energies according to the rotations

•

Plotted rotations•Picture on the right: energy vs. rotations plot•Came up with the same 3 low energy configuration (alpha helix, beta sheet, random coil)•

Protein Secondary Structure (2° Structure)

α-helix.-

β-sheet, β-turn.-

Random coil.-

Adjacent amino acids typically interact through noncovalent interactions to form 3-D structures:•

Stable 3D conformation resulting from interactions between adjacent amino acids in the primary structure•

The Alpha HelixProtein Secondary Structure•

Biochem 1 - Test 2

FIGURE 4-4 Models of the α helix, showing different aspects of its structure. (a) Ball-and-stick model showing the intrachain hydrogen bonds. The repeat unit is a single turn of the helix, 3.6 residues. (b) The α helix viewed from one end, looking down the lon gitudinal axis (derived from PDB ID 4TNC). Note the positions of the R groups, represented by purple spheres. This ball-and-stick model, which emphasizes the helical arrangement, gives the false impression that the helix is hollow, because the balls do not represent the van der Waals radii of the individual atoms. (c) As this space-filling model shows, the atoms in the center of the α helix are in very close contact. (d) Helical wheel projection of an α helix. This representation can be colored to identify surfaces with particular properties. The yellow resi dues, for example, could be hydrophobic and conform to an interface between the helix shown here and another part of the same or another polypeptide. The red and blue residues illustrate the potential for interaction of negatively and positively charged side chains separated by two residues in the helix.

If you draw 3D backbone as a ribbon the alpha helix will look like figure A ○

Peptide backbone structure: looking at the location in space of alpha amino groups, alpha carbons, alpha carbonyls -

Has an axis of symmetry-

Alpha helix -

If you rotate and helix and look down the axis of symmetry you can see the location of the substituent groups-

This means that when the amino acids are about 4 apart in the alpha helix, they can interact with one another - can see each other

○

Hydrogen bonding (between alpha amino and alpha carbonyl groups within the backbone)○

Stabilization of alpha helix○

Called a spiral or solenoidal coil○

The residue is about 4-

Stabilization of alpha helix: Intra-helical hydrogen bonding between alpha nitrogen and alpha carbonyl in peptide backbone that are four residues apart1)Attractive interactions between R-groups four residues apart stabilize alpha helix2)

Amino terminus instead of taking helical structure will unwind looking for a more stable lower energy conformation

R group with positive charge at amino terminus : destabilize the helix○

Nonpolar groups at the termini have no effect ○

Resonance of positively charged R group at carboxyl terminus stabilizes helix. Negatively charges group at N-terminus (amino terminus) stabilizes helix

3)

Figure A:•

Stuck on the outside to interact with the environment ○

Dark spots = R group-

Interior of alpha helix Grey middle = backbone-

Figure B:•

Biochem 1 - Test 2

Interior of alpha helix ○

Backbone shielded from interactions with water because R groups are on the outside ○

Look at location of the atoms○

Space filling model-

R groups on the outside -

Interaction between substituent groups on outside of the helix stabilizes the helix-

Figure C:•

The 1st substituent is positively charged and the 4th is negatively charged so they react in an attractive interaction-

Causes helix to unwind and look for more stable conformation ○

If they were both negative they would react in a repulsive interaction (destabilizing) -

4 interacts with 7 -

Figure D:•

Why? - because the two sides don't see each other○

So you can have ion channels going through the membranes - perfectly happy in the membrane and outside of the membrane

Important because you can have proteins that insert into the cell membrane and the nonpolar part of the alpha helix is happy to interact with the fatty inside of the cell membrane while the polar part is happy to interact with the ions in an aqueous environment

○

Based on direction/interactions of R groups in alpha helix ○

Can be amphipathic (R group on one side is polar and the R group on the other side is nonpolar)-

Physical property:•

Location of R-Groups: Physical and Stabilizing Properties

Intra helical hydrogen bonding stabilizing alpha helix•

Since the peptide bond is polar and the amino groups are not hydrogen bonded, the amino terminus has a slightly positive charge (and that is also why the carboxyl terminus has a slightly negative charge)

-

First 2-3 alpha amino groups at each end do not hydrogen bond•

If we find an alpha amino that is negatively charged in the first 2-3 residues in the amino terminus, the negative charge will stabilize the partial positive charge - that stabilizes the helix

•

α-Helices May Be Right- or Left-Handed

In the picture to the left, the top of the coil (away from the hand) is the N-T•

Lower energy-

Right handed helix is predominant in biological system •

Biochem 1 - Test 2

Lower energy-

More stable-

The middle pole is the peptide backbone -

Picture above on the right = right handed alpha helix

If you have to rotate it clockwise = right handed alpha helix○

Counterclockwise = left handed alpha helix ○

Which way do you have to rotate the screw to drive it into the ground (sink the thread)?-

Picture on the left, imagine there is a screwdriver and the spirals are the pads of the screw•

The Beta SheetProtein Secondary Structure•

Take paper, fold into pleats, unfold: beta strand•

They come in beta sheets-

You never find a beta strand alone in nature•

Friday, October 14, 2011

Beta sheet (top view)•

This pairing = antiparallel-

(Pair amino terminus with carboxyl terminus) -

In each strand, the carbonyl end is paired with the amino of the other•

Carboxyl = slightly negative, amino = slightly positive -

Head of arrow = carbonyl, tail = amino•

These strands are usually a part of a larger protein and there isn't really an amino terminus •Above picture is how you draw out representation of amino strands •

(Different from an alpha helix where you have intra helical hydrogen bonding)

There is hydrogen bonding between strands - between the amino and carbonyl groups in adjacent strands○

There is stabilization between R groups as well○

Weak electrostatic interactions that depend on distance

R groups on the top of the sheet don't interact with R groups on the bottom of the sheet○

Inter strand hydrogen bonding between amino and carbonyl groups in peptide backbone-

Beta sheets can be amphipathic (the tops can be nonpolar and the bottoms can be polar and everything is fine)○

Top R groups don't see bottom R groups○

Pretty stable : straight line hydrogen bonding ○

Something slightly positive interacting with something slightly negative = stabilizing ○

Attractive interactions between R groups adjacent in space-

Stabilization:•

FIGURE 4-6a The β conformation of polypeptide chains. These top and side views reveal the R groups extending out from the β sheet and emphasize the pleated shape described by the planes of the peptide bonds. (An alternative name for this structure is β-pleated sheet.) Hydrogen-bond cross-links between adjacent chains are also shown. The amino-terminal to carboxyl-terminal orientations of adjacent chains (arrows) can be the same or opposite, forming (a) an antiparallel β sheet or (b) a parallel β sheet.

Biochem 1 - Test 2

All of the amino ends interact with each other, all the carbonyl ends interact with each other•

No straight hydrogen bonding (weaker bonds)-

Less stable than antiparallel•

All of the negatively charged carbonyl groups are repulsive-

Causes end of sheets to twist-

Twisting results in straight line hydrogen bonding-

All of the strands added together = a beta sheet•

Twisting results in off straight line hydrogen bonding -

Twisted because of the repulsion from the amino and the carboxyl terminal of the individual strands of the sheet •

β-Turn, Ω-LoopProtein Secondary Structure•

The two pictures above are both beta turns•Don't worry about differentiating between type one and two•

Beta turns : puts rapid change in direction of a protein-

General characteristics:•

FIGURE 4-6b The β conformation of polypeptide chains. These top and side views reveal the R groups extending out from the β sheet and emphasize the pleated shape described by the planes of the peptide bonds. (An alternative name for this structure is β-pleated sheet.) Hydrogen-bond cross-links between adjacent chains are also shown. The amino-terminal to carboxyl-terminal orientations of adjacent chains (arrows) can be the same or opposite, forming (a) an antiparallel β sheet or (b) a parallel β sheet.

FIGURE 4-7a Structures of β turns. (a) Type I and type II β turns are most common; type I turns occur more than twice as frequently as type II. Type II β turns usually have Gly as the third residue. Note the hydrogen bond between the peptide groups of the first and fourth residues of the bends. (Individual amino acid residues are framed by large blue circles.)

Biochem 1 - Test 2

Glycine : is the smallest R group (anything that is bigger won't allow you to have a β-turn○

Only amino acid that forms stable cis peptide bonds

Proline : forms stable cis peptide bonds○

99% of β-turns will have proline and glycine as its residue-

Consists of 4 amino acid residues•

This is a solution so things can jus as easily get out of β-turn as form them•β-turn is stabilizes by hydrogen bonding between alpha amino and alpha carbonyl of the 1st and 4th residues•

A = beta turn•

Bigger structure-

B = omega loop (can contain 8-16 amino acids)•

Circular Dichroism Spectroscopy (CD Spectroscopy)

Looking at the difference in rotation of plane polarized light•Look at a variety of wavelengths and you get s spectrum of differences and you will have a characteristic spectrum for alpha helices, beta sheets, and random coils conformations

•

You can follow changes of secondary structure under different conditions•

Protein Tertiary Structure (3° Structure)Stable, compact, biologically functional 3D conformation of a protein•Result from long-range amino acid interactions (don't have to be adjacent for interactions to occur)•The protein collapses on itself •

Omega loops (beta loop) consist of 6 to 16 amino acids. Can be viewed as a short stretch of random coil structure.

Measures absorbance of polarized, far UV light (190-250nm) by the peptide bond. The difference in absorbance of polarized UV light (∆ε) is due to changes in the folding of the peptide backbone. CD is used to determine secondary structure and can be used to follow protein folding or denaturation.

Biochem 1 - Test 2

As it collapses, amino acid residue interactions determine the final conformation-

(Secondary structure - adjacent amino acids rotate past each other to determine secondary structure) -

The protein collapses on itself •

Size of Human Serum Albumin in Various Conformations

FIGURE 4-15a,b Tertiary structure of sperm whale myoglobin. (PDB ID 1MBO) Orientation of the protein is similar in (a) through (d); the heme group is shown in red. In addition to illustrating the myoglobin structure, this figure provides examples of several different ways to display protein structure. (a) The polypeptide backbone in a ribbon representation of a type introduced by Jane Richardson, which highlights regions of secondary structure. The α-helical regions are evident. (b) Surface contour image; this is useful for visualizing pockets in the protein where other molecules might bind. (c) Ribbon representation including side chains (blue) for the hydrophobic residues Leu, Ile, Val, and Phe. (d) Space-filling model with all amino acid side chains. Each atom is represented by a sphere encompassing its van der Waals radius. The hydrophobic residues are again shown in blue; most are buried in the interior of the protein and thus not visible.

Tertiary structure•The picture above are of myoglobin•Purpose = oxygen storage•8 helix bundle•

Look at the surface (you can't tell it is an 8 helix bundle)-

The heme group binds the oxygen○

The heme is located in hydrophobic valley on the surface of myoglobin-

B is a space filling model•

To decrease the size to fit into our body -

A lot of compaction with tertiary structure -

Forming of tertiary structures - compact stable form-

Why do we need this compact structure?•

FIGURE 4-15c,d Tertiary structure of sperm whale myoglobin. (PDB ID 1MBO) Orientation of the protein is similar in (a) through (d); the heme group is shown in red. In addition to illustrating the myoglobin structure, this figure provides examples of several dif ferent ways to

Biochem 1 - Test 2

heme group is shown in red. In addition to illustrating the myoglobin structure, this figure provides examples of several dif ferent ways to display protein structure. (a) The polypeptide backbone in a ribbon representation of a type introduced by Jane Richardson, which highlights regions of secondary structure. The α-helical regions are evident. (b) Surface contour image; this is useful for visualizing pockets in the protein where other molecules might bind. (c) Ribbon representation including side chains (blue) for the hydrophobic residues Leu, Ile, Val, and Phe. (d) Space-filling model with all amino acid side chains. Each atom is represented by a sphere encompassing its van der Waals radius. The hydrophobic residues are again shown in blue; most are buried in the interior of the protein and thus not visible .

Both of the picture above are myoglobin•

A lot of nonpolar R groups in myoglobin-

Tertiary structures - there are nonpolar R groups on the interior of the protein -

C = blue = nonpolar amino acid R groups•

Tertiary structure buries nonpolar R groups on interior of the protein•X-ray crystal structures•

Gain of entropy and water○

Hydrophobic effect-

What drives nonpolar R groups to the interior?•

X-Ray Crystallography is Used to Determine 3° Structure

Steps in determining the structure of sperm whale myoglobin by x-ray crystallography. (a) X-ray diffraction patterns are generated from a crystal of the protein. (b) Data extracted from the diffraction patterns are used to calculate a three-dimensional electron-density map. The electron density of only part of the structure, the heme, is shown. (c) Regions of greatest electron density reveal the locat ion of atomic nuclei, and this information is used to piece together the final structure. Here, the heme structure is modeled into its elec tron-density map. (d) The completed structure of sperm whale myoglobin, including the heme (PDB ID 2MBW).

Attached to one amino acid residue in the myoglobin-

Attached to myoglobin by HISTIDINE-

Heme group has iron in it•

Nitrogen bonds to the iron by covalent bonds-

This breaks the bond and frees the heme group-

Iron has an empty d-orbital•

Once you get a crystal you can bombard it with x-rays-

The free heme group then crystallizes •

X-rays reflect off electron cloud of heme group•The top left corer picture = diffraction pattern •

Protein Structural MotifsSupersecondary Structure•Complex, stable 3D conformations formed by association of secondary structures•

Structural Motifs Can be Simple or Complex

(a) A simple motif, the β-α-β loop. (b) A more elaborate motif, the β barrel. This

Biochem 1 - Test 2

β-α-β loop = parallel β sheet (you can see the twisting)•

Anti parallel-

Toxin that destroys bacteria by forming a pore in the bacteria-

Can reside in bacterial membrane-

One face is hydrophobic (stable interaction with membrane)-

The other face is hydrophilic (allows electrolytes and things to come in)-

β-barrel = random coils holding it together•

Adding structural complexity•

Domains in Proteins

Domains often have specific functions within a protein such as binding small molecules.

Independently folded region of a protein - usually contains super secondary structures-

Each lump is an alpha beta barrel-

Final structure of protein depends on amino acid residue sequence and how everything interacts with each other -

Structural domains: if you cut them apart they will stay in a alpha beta barrel ○

If each weren't independently folded, if you cut the two apart, they would unwind - no longer in an alpha beta barrel-

If you cut the two lumps apart anywhere besides where they join (cut one in half) - it will fall apart -

Structural domain:•

Amino acid residues that perform a function in a protein-

Amino acid residues in the protein that bind to dopamine = binding domain ○

Dopamine receptor-

Enzyme at the site (binds protein and catalyzes reaction) will have two functional domains (binding and analytical)-

If you cut them apart, they unfold-

Functional domains:•

Monday, October 17, 2011

Protein Quaternary Structure (4° Structure)A single, functional protein forms from individual proteins.•Highest level of structure•Final, stable, biologically functional 3D conformation of a single protein made-up from the association of 2 or more other proteins•

Structural domains in the polypeptide troponin C. (PDB ID 4TNC) This calcium -binding protein associated with muscle has two separate calcium-binding domains, indicated in blue and purple. A domain is defined as a part of a polypeptide chain that is independently stable or could undergo movements as a single entity. Domains can be either be easily distinguished, as above, or be obscured by extensive contacts with other domains or parts of the protein.

FIGURE 4-20 Constructing large motifs from smaller ones. The α/β barrel is a commonly occurring motif constructed from repetitions of the α-β-α loop motif. This α/β barrel is a domain of pyruvate kinase (a glycolytic enzyme) from rabbit (derived from PDB ID 1PKN).

(a) A simple motif, the β-α-β loop. (b) A more elaborate motif, the β barrel. This β barrel is a single domain of α-hemolysin (a toxin that kills a cell by creating a hole in its membrane) from the bacterium Staphylococcus aureus (derived from PDB ID 7AHL).

Biochem 1 - Test 2

These other proteins = subunits-

Final, stable, biologically functional 3D conformation of a single protein made-up from the association of 2 or more other proteins•

You have a single biologically functional protein that is made up from other proteins (subunits)•

Tertiary vs. Quaternary Structure

Oxygen storage-

Monomeric - one form-

The picture on the left = myoglobin (mb)•

Oxygen transport-

Each subunit kind of looks like myoglobin (has 2 different types of subunits)○

2 of the 4 subunits are different from each other○

Single protein with four subunits -

Many forms (oligomeric)-

Oligomeric protein because it has quaternary structure -

α2β2 = 2 pairs of 2 different subunits-

Each subunit has its own tertiary structure○

Have to have super secondary structure? NO

Have to contain structural domains? NO

Have to have secondary structure? YES

Have to have primary structure? YES

Even if they don’t have α helices or β sheets, random coil is still secondary structure○

Protein with 4 subunits - 2 pairs of 2 different subunits -

The picture on the right = hemoglobin (hb)•

6 subunits (3 different types of subunits)-

If the subunit composition is α2βγ3 - it is oligomeric•

Crystal structure of hemoglobin•Heme group (oxygen binding group) is buried in a valley in the hemoglobin•

Biochem 1 - Test 2

Hydrophobic valley-

Heme group (oxygen binding group) is buried in a valley in the hemoglobin•

Protein FoldingFormation of Functional Proteins•Tertiary structure result from a protein folding - looking for stable conformations •

Thermodynamics of Protein Folding

Enthalpy of H-bonds is similar.-

Overall ∆H is slightly negatively charged○

Enthalpy of protein slightly decreases due to stabilizing London forces.-

Entropy of folded protein decreases.-

Overall ∆S is slightly positively charged ○

Entropy of water increases when protein is folded.-

UnFolded vs. Folded•

High energy, high entropy states-

Folds into tertiary structure○

As the protein folds - becomes more stabilized through electrostatic interactions -

The top of the picture = denatured/unfolding•

Low energy, low entropy states-

The bottom of the picture = folded (tertiary structure)•

When a protein folds it falls down in ∆G (energy) and ∆S (entropy) well•

Water hydrogen bonds with water-

After protein folds - polar residues from protein interact with each other or water ○

Enthalpy - potential chemical energy that is held in the stabilization of formation of chemical bonds ○

Enthalpy of hydrogen bonding interactions before and after a protein folds = similar-

In unfolded state → all of the polar molecules in the protein are either interacting with each other or with water•

Protein experiences huge drop in entropy - but water (because there is so many more molecules of water than protein) will experience a slightly larger gain in entropy

□

So ∆S = slightly positive□

Why is exclusion thermodynamically favorable? - the entropy of water goes up (released from hydration cages)

Hydrophobic effect happens → water excludes nonpolar molecules → proteins fold so nonpolar residues' interactions with water is minimized (they are folded into the interior of the protein)

○

It has nonpolar residues exposed to water (this is non favorable)-

When nonpolar residues are folded into interior of protein → they become closely packed together-

What happens when a protein fold?•

Biochem 1 - Test 2

Strength of London forces between nonpolar molecules goes way up○

London dispersion forces between nonpolar molecules = stabilizing, energy of protein goes down○

When nonpolar residues are folded into interior of protein → they become closely packed together-

Overall, ∆H = slightly negative-

When protein fold - low entropy○

When in solution - high entropy-

Protein is losing a lot of entropy but there are many more water molecules so the gain in entropy by water is slightly largerthan the loss of entropy of the protein

○

∆S = slightly positive -

Overall ∆G = slightly negative -

∆G = ∆H - T∆S•

Stages of Protein Folding

You start with an unfolded state then start forming secondary structure-

A and B in the picture can rotate and switch between α helix and β sheets○

Because protein is rotating about ɸ and ψ looking for low energy conformations○

You can form an alpha helix or beta sheet-

Formation of super secondary structures-

Picture on the left:•

You form super secondary structure → they will hit an unstable conformation and fall apart-

Fall apart into secondary structures-

Secondary structures fall apart and can reform and try to reform super secondary structure (all based on low energy conformation)-

You end up forming domains then tertiary structures-

Picture in the middle•

The phase between an unfolded protein and its final tertiary structure = molten globule-

Constantly changing looking for lowest energy conformations○

It is not necessary that super secondary structures and domains form○

It is not necessary that α helices or β sheets form○

You will have random coil that will associate with itself into a compact ball○

Molten globule = changeable -

Picture on the right:•

Stable structure for this protein = misfolded protein -

Misfolding of proteins often leads to various pathophysiology (Alzheimer's) -

When a protein is folding it can get trapped in one of the alternative low energy conformations •

Protein Folding is Driven by the Hydrophobic Effect

Biochem 1 - Test 2

Look at where residues are-

If you look a protein and separate its amino acid residues based on whether its hydrophobic or hydrophilic •

Hydrophilic = on exterior -

You can prove that hydrophobic = on interior•

Protein folding is driven primarily on hydrophobic effect•

Thermodynamics of Protein Folding

Both = chaotropic agents •Both used to unfold proteins •

When you put them in water, water prefers to H-bond with them rather than other water molecules - you break the hexagonal array-

↑ ∆S (increase entropy)○

When you break hexagonal array and go to something random: -

Very strong hydrogen bonders•

Before the nonpolar residues were excluded, water is trapped in hydration cages in order to H-bond○

When nonpolar residues are isolate, water moves back out of hexagonal array and goes around○

Entropy increases by a lot ○

Structure of water is now completely random so it doesn't care is nonpolar residues are isolated or not○

Exclusion of nonpolar residue from water's structure increases the entropy in water-

Why did water exclude nonpolar residues from its structure in the first place?•

Guanidinium ion = 6M in order to unfold a protein•Urea = 8M in order to unfold a protein•

Overall ΔG for folding is only slightly negative.-

Unfolded vs. Folded•

Folded structure of protein (tertiary structure) is only slightly stable.-

(Breathability)○

Important for protein function and physiological function○

All a result of slight negative free energy folding ○

Protein has conformational flexibility.-

Result:•

∆G is slightly negative (for overall folding process)-

∆S goes up slightly on folding-

∆G = ∆H - T∆S•

Myoglobin•

Structure in a protein is shifting by a little -

Picture is blurry because protein has conformational flexibility •

Biochem 1 - Test 2

Another example = heat (when you heat up something you are supplying energy - creates greater molecular motion)○

These two increase entropy = chaotropic angents-

Urea = 8M in order to unfold a protein•

Role of Disulfide Bonds in Formation of Tertiary Structure

Disulfide bonds are important in the tertiary structure•Amino acid involved in the formation of disulfide bonds = cysteine•Above you have a native, active protein and you throw in 8M urea •

Thiol○

Takes disulfide bonds (-S-S-) which are oxidized and reduce them into the free sulfhydryl (-SH HS-)○

Mercaptoethanol (or oithiothreitoc) = reducing agent-

Break up all the disulfide bonds - protein no longer has to isolate nonpolar residues on interior → protein unfolds into denatured state

-

STEP 1•

Disulfide bonds return/reform○

Remove mercaptoethanol (but leave in 8M urea)-

STEP 3•

Remove urea and let protein refold = stable 3D conformation this is inactive because the disulfide bonds form between the wrong cysteines

-

When you remove the urea the protein folds into its stable structure but it is not its native, biologically active structure -

STEP 4•

Put in mercaptoethanol then remove it - disulfide bonds should properly reform = active protein-

STEP 5•

99.95% of proteins, when you denature it and try to put it back in its native structure, doesn't work •

Correct Protein Folding May Require Chaperones

Renaturation of unfolded, denatured ribonuclease. Urea denatures the ribonuclease, and mercaptoethanol (HOCH2CH2SH) reduces and thus cleaves the disulfide bonds to yield eight Cys residues. Renaturation involves reestablishing the correct disulfide cross-links.

Biochem 1 - Test 2

Some proteins can fold correctly on their own (small amount)-

What do we need to have correct protein folding?•

Chaperones : proteins that give help to correct protein folding•

Help form disulfide bonds properly○

Break and reform disulfide bonds ○

Protein disulfide isomerase (PDI)-

Peptide prolyl cis-trans isomerase (PPI)-

Prevent aggregation○

Act as folding template○

Molecular chaperones-

"Water bomb"○

Huge proteins ○

Chaperonins-

Classes of chaperones:•

Kinetically trapped = fall into energy well -

Denature protein then cool = inactive protein•

If you put it in presence of other chaperone proteins = native active protein •

Wednesday, October 19, 2011

Biochem 1 - Test 2

Break disulfide bond, then oxidize -

PDI : reduce disulfide (break) then oxidize in another place (form in another location)•

Misfolded protein - protein disulfide isomerase comes in (it has 3 thiol groups) - 3 thiol groups are used to break up the disulfide bond

-

Positions of the bonds are shifted to 1 + 4 and 2 + 3-

Protein has disulfide bonds between positions 1 + 2 and between 4 + 3•

Big group of nonpolar residues on the surface of the proteins usually is an indication-

How does PDI know that the disulfide bonds are in the wrong place? - we do not know•

Globular protein with large amount of nonpolar residues on surface is a tip off to chaperon proteins that something is wrong and it needs to be refolded

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If a lot of nonpolar residues are on the outside, PDI will bind to it and shift around the disulfide bonds → protein has ability to refold into another conformation that may bury hydrophobic residues more

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Globular protein folds by hydrophobic effect - putting hydrophobic amino acid residues on interior of protein•

PPI = Peptidyl prolyl isomerase -

Commonalities of when it occurs - large amount of nonpolar residues on the surface of the protein○

PPI binds the protein, switches around a few bonds → gives different rotational possibilities for the protein to fold ○

How does PPI know when it needs to change a conformation from trans to cis or cis to trans? - We do not know-

PPI : changes (conformation) stereochemistry of peptide bonds involving proline •

Molecular chaperons

Biochem 1 - Test 2

FIGURE 4-29 Chaperones in protein folding. The cyclic pathway by which chaperones bind and release polypeptides is illustrated for the E. coli chaperone proteins DnaK and DnaJ, homologs of the eukaryotic chaperones Hsp70 and Hsp40. The chaperones do not actively promote the folding of the substrate protein, but instead prevent aggregation of unfolded peptides. For a population of polypeptide molecules, some fraction of the molecules released at the end of the cycle are in the native conformation. The remainder are rebound by DnaK or diverted to the chaperonin system (GroEL; see Figure 4-30). In bacteria, a protein called GrpE interacts transiently with DnaK late in the cycle (step 3), promoting dissociation of ADP and possibly DnaJ. No eukaryotic analog of GrpE is known.

Don't need to memorize•

If you have unfolded protein with a large amount of nonpolar residues on the surface - if two meet up by hydrophobic effect they will be driven together (aggregate)

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Proteins will meet in nonnative conformations - can cause serious problems -

Step 1 : prevent aggregation of unfolded proteins•

If they bind to one part of a protein - they lock that protein and prevents it from rotating in one part (rest of protein is free to rotate around)

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Step 2 : act as a template to direct folding •

Okay if folded protein is a small protein (20,000 MW)○

As you get larger, the molecular chaperones are a stepping system and you end up having a partially folded protein ○

Partially folded protein goes to GroEL system (chaperone)

Partially folded protein still has a lot of nonpolar residues on its surface○

One result can be action of molecular chaperone results in directly folded protein and they unbind-

Step 3 : results•

GroEL = chaperonin •

Biochem 1 - Test 2

FIGURE 4-30a Chaperonins in protein folding. (a) A proposed pathway for the action of the E. coli chaperonins GroEL (a member of the Hsp60 protein family) and GroES. Each GroEL complex consists of two large pockets formed by two heptameric rings (each subuni t Mr 57,000). GroES, also a heptamer (subunit Mr 10,000), blocks one of the GroEL pockets. (Text and picture are from different books.)

Multi subunit protein - quaternary structure -

Chaperonin = oligomeric protein •

GroEL•Chambers : ATP binding sites (you don't need to know where the ATP binds or how many binds)•It has many shapes •

So it binds to ATP → hydrolyzes the ATP to ADP → this gives it the energy to go through conformational changes -

Monster protein that is undergoing many conformational changes and it needs energy to drive the changes •

It is globular - a lot of nonpolar residues on the surface○

Therefore the groEL knows it isn't right and needs to try to be folded together○

How does it bind/how does groEL know to bind this? - there is a large number of nonpolar residues on the surface of the protein-

When groES binds to groEL → causes large conformational change in groEL (unfolded protein is bound)○

Once the unfolded protein binds to groEL, it alters the conformation of groEL a little → one of the other subunits (groES) recognizes groEL's binding site and groES binds to groEL

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When groES binds to groEL there is a large conformational expansion and unfolded protein is dropped into the chamber of groEL -

Unfolded protein in the picture above → binds to one chamber of the groEL complex •

When unfolded protein binds to the empty chamber, groES pops off and the folded protein is spit out of that chamber (then theprocess repeats)

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If it is ok then it is considered to be folded and that is the end of the story ○

Protein that is spit out is better folded (if it still has too many nonpolar residues on the surface it will rebind to the chaperonin and restart the process again)

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Interaction between groES and groEL causes another interaction → unfolded protein binds to chamber•

Biochem 1 - Test 2

FIGURE 4-30b Chaperonins in protein folding. (b) Surface and cut-away images of the GroEL/GroES complex (PDB ID 1AON). The cut-away (right) illustrates the large interior space within which other proteins are bound.X-ray crystal structures of a chaperonin •

When the chamber expands, suddenly a lot of water appears on the walls of the chamber○

(All the white holes represents water molecules)○

In the chamber, before it expands, the atmosphere of the chamber is primarily nonpolar -

Water tightly bound to interior-

Puts protein with a lot of nonpolar substances on surface in an intensely polar environment → forcing hydrophobic effect on protein

Drives nonpolar residues to interior of protein

When it expands → water comes to the surface and generates a water bomb ○

When chamber is contracted → water is concealed-

When chamber expands it forces water to surface of chamber = puts it in a polar environment -

This is going on all the time in our cells -

In the picture to the right (above):•

Has directionality•White spots = H2O•

Fibrous ProteinsStructure Yields Function•

Biochem 1 - Test 2

Structure Yields Function•Function: structural•Property: hydrophobic by nature•

When located properly in cell → peptides = cleaved off (and is hydrophobic)○

Like pro collagen - has extra bits of peptide on it that makes it water soluble-

Synthesized as a pro protein •

Structure of Silk

Hair, Fingernails, Claws, and Hooves

All of these = made out of protein keratin•

Primarily nonpolar amino acids in primary structure -

Exists as a coil of two α helices of keratin together-

Keratin is made as a single α helix (right handed)•

There is repulsion at both ends -

Picture 2: left side = amino terminus, right side = carboxyl•

Protofilament = stabilized by:-

Picture 3: 2 helical coils of keratin associates with 2 other helical coils = protofilament •

Silk threads are hard to break-

Silk = antiparallel beta sheets stacked on top of each other

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Silk: •

One pair of sheets only has alanine-

The other only has glycine interacting with each other

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These sheets are just held together by London dispersion forces (weakest force known)

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What are the interactions between the stacked sheets?

•

When you sum the chemical potential energy in all the London forces you get a very strong structure

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Alanine and glycine = very small (not a lot of dispersion forces between them)

•

Biochem 1 - Test 2

Electrostatic interactions (carboxyl and amino termini interact between the coil helices) ○

Keratin has a lot of cysteine residues - coils are further held together by disulfide bonds ○

Protofilament = stabilized by:-

Disulfide bonds within the protofilaments and disulfide bonds between the protofilaments in the protofibril -

Picture 4: 2 protofilaments come together to make a protofibril •

All helices of keratin = linked together by disulfide bonds •

Nails = more stable so it is more rigid ○

Hair has more disulfide bonds than fingernails - less rigid -

Number of disulfide bonds makes a difference in physical properties of molecules -

What gives hair, fingernails, claws and hooves their different properties? - the amount of disulfide bonds•

Α helix → coil → protofilament → protofibril •Protofibrils = bundled together to form the intermediate filament•Intermediate filaments form the cell•

So, do you want relaxed or tight curls from your perm?

If you want curly hair you have-

Mercaptoethanol/dithiothreitol is used to reduce the disulfide bonds and break them at the sulfhydryl -

Straight hair → disulfide bonds are in line•

If you reoxidize you form the straight hair again •

Small roller = a lot of misalignment in disulfide bonds -

Put hair in roller and then oxidize = curly hair -

Hair in large roller - you physically alter the conformation and misalign them a little•

To straighten hair, you have to break the disulfide bonds, align them to be straight and then reoxidize -

It is easier to curl hair then to straighten it•

Why does hair get frizzy in the humid weather? - conformation depends on hydration •

FIGURE 4-10b Structure of hair. (b) A hair is an array of many α-keratin filaments, made up of the substructures shown in (a).

Biochem 1 - Test 2

The Three-Dimensional Structure of Proteins - Chapter 4

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