Protein StructuresI. Thermodynamic and evolution points of view for protein synthesis

Amino acids are polymerized into polypeptide chain on ribosomes in the cell. The chain direction is defined from the amino end (N-terminus) to the carboxyl group (C-terminus) corresponds to the 5’ to 3- direction on the messenger RNA.

(a) Most free energy is spent in peptide bond formation for accuracy in protein translationOverall yield of free energy per peptide bond formation: 25 kcal/mole�G per Peptide bond formation in water : 5 kcal/mole

(b) lowest cost bond allow fast adaptation to environmental changessynthesized peptide bond at low expense, better protection of itself from less favorable environmental changes.

Structural characteristics of peptide bonds and its implications

I: free rotation of C’-N bond

II: planar, large dipole moment, inhibit rotation

The hybrid structure contains 60% of I and 40% of II, as derived from the C’-N bond length. The resonance energy is 85 KJ/mole. Structure I: Structure II = 3:2 where the �-electrons smear over C’-O and C’-N bonds. Therefore, Ci

�, Ci’, Oi, Ni+1, H i+1, C�

i+1

The stiff peptide bond restricts the polypeptide chain flexibility.The peptide unit is rather bulky and gives rise to substantial steric hindrance.

The conformation of peptide group

The C’-N bond is 0.13 Å shorter than N-C� bond.The C=O bond is 0.02 Å shorter than that of aldehydes and ketones.

Peptide groups prefer the trans conformation. The successive C� atoms are on the opposite sides of the peptide bond joining them. The cis conformation is ~8 kJ/mole less stable than the trans conformation.

Definition of �, ����, torsion angles

protein orientationThe conformation of the polypeptide chain is essentially described by the dihedral angles � and atthe C�-atoms as shown in the figure.�i=0 for Ci

�_ Ci’ trans to Ni_ H i

�i=0 for Ci�_ Ni trans to Ci

’- Oi�i=0 for Ci

�_ Ci’ cis to Ni+1

_ C�i+1

The planarity of the peptide bond restricts omega to 180 degrees in very nearly all of the main chain peptide bonds.i=0 for Ci

�_ Ni cis to Ci�_ Oi

�

i define the conformation of the amino side chain

You always view from the N-terminal side.

Ramanchandran PlotExplore possible N-C� bond and C�-C’ bond free rotation region.

Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii Therefore, phi and psi angles which cause spheres to collide correspond to stericallydisallowed conformations of the polypeptide backbone.

(back)

Protein �, angles distribution

The red regions correspond to conformations where there are no stericclashes, ie. these are the allowed regions namely the alpha-helical and beta-sheet conformations. The yellow areas show the allowed regions if slightly shorter van derWaals radii are used in the calculation, ie the atoms are allowed to come a little closer together. This brings out an additional region which corresponds to the left-handed alpha-helix.

The 3(10) helix occurs close to the upper right of the alpha-helical region and is on the edge of allowed region indicating lower stability.

The white areas correspond to conformations where atoms in the polypeptide come closer than the sum of their van der Waals radii.

Ramanchandran Summary*

Except for glycine and proline, all sterically allowed regions are essentially the same.

(i) (-60º, -60º) region reflects right-handed-helix positionwhereas (+60º, +60º) is for left-handed-helix.

(ii) (-90º, +120º) corresponds to extended chain and to residues in �–sheets.

(iii) figure 8-7, 5% of protein residues analyzed with C�-atom lie in a forbidden region.

(iv) Proline behaves differently because side chain binds to the peptide nitrogen and fixes to –60º 20º.

Cis or trans conformation

(b) Proline is the only residue most susceptible to cis bondsformation.*

(a) peptide synthesis in ribosome is stereospecific, either -cis or –trans.

It is unlikely that a specific conformation arise from(i) post-translational isomerization because many specific enzyme

will be required(ii) spontaneous isomerization during folding process because of the

energy barrier.

Cis-alanine, (-155�, +105�)

Peptide bond and peptide plan

deviation in a bond angle � 5ºdeviation in a bond length � 0.05Ådeviation in torsion angle � 12ºcorresponds to a potential energy increase of 1 kcal/mole.

Plasticity arise from(a) non-planar peptide bond.(b) the conformational space at one residue is multidimensional.(any slight hindrance can be circumvented by small deviations in bond and torsion angles as well as in bond lengths.)

� � angles peptide plane peptidebond

Secondary structure: �-Helical parameters

r = radius of the helix** d is positive, helix chiralitycan be positive or negative.Notation:nm: n = residues per turn andm = number of atoms, including H, in the ring closed by H-bonding.

n = residues per turn. (n rarely is interger in protein.)d = axial shift (rise) per residuen.d = pitch of the helix

Helices are the stabilizing elements of protein structure. The polypeptide chain is twisted by the same amount about each of its C� atoms to form a helical conformation.

Structural characteristics of �-Helix

(1) hydrogen bonding between peptide C=O and (i+4)th residue N-H (2.8Å)

(2) core of the �-Helix is tightly packed.(3) R-group pointed backward or outward

from the helix.very stable and most abundant. (In globular proteins, average length of the �-Helix is about ~12 a.a., corresponding to 18Å residues or 3 helix turns.)

(4) The torsion angle �= -57º, and =-47 º, n=3.6 residue per turn, and a pitch of 5.4 Å.

Successive peptide units assume identical orientations, that is the (�,) angles are the same, the polypeptide backbone forms a linear group. Helix has chirality; it may be either right handed or left handed)

�-Helix

• dipole forming the H-bonds are aligned (in the minimum energy)

• side chain packing favorable well staggered.

• helix radius r allows for van der Waals attraction across the helical axis. (Kink of 20are often produced by incorporating pro residue)

The � helical polypeptide conformation has the simultaneous allowed conforma-tionangles and a favorable H-bonding pattern.

310-Helix

• dipole forming the H-bonds are not aligned (not in the minimum energy).

• Side chain packing on identical positions (unfavorable).

• Rare (usually one turn) and tend to be at the N- and C-termini. Short segments of 3.10 helix are found. The ends of alpha helices can contain a single 3.10- or pi-like hydrogen bond.

�-Helix• Dipole forming the H-bonds are

reasonably aligned which forms a hole inside because of its large radius r.

• side chains are staggered better than those in 310-Helix.

• one turn of �-helix is sometimes found at the ends of regular alpha helices but pi helices longer than a few i, i+5 hydrogen bonds are not found.

The infrequency of �-helix• The phi and psi angles of the pure �-helix (-57.1, -69.7) lie at the very edge of an

allowed, minimum energy region of the Ramachandran (�, �) map.• The �-helix requires that the angle �(N-C�-C') be larger (114.9) than the standard

tetrahedral angle of 109.5 degrees.• The large radius of the �-helix means the polypeptide backbone is no longer in van

der Waals contact across the helical axis forming an axial hole too small for solvent water to fill.

• Side chains are more staggered than the ideal 310 helix but not as well as the alpha helix.

Different types of �-HelixLinear group observed residues per

turn n and chirality

rise per residue,d (Å)

radius of helix r (Å)

�-Helix(right-handed)(Notation:3.613)

Abundant +3.6 1.5�= -57 , = -47

2.3

�L-Helix(left-handed)(Notation: 3.613)

Hypothe-tical

-3.6 1.5�= +57 ,= + 47

2.3

310-Helix (310) Smallpieces

+3.0 2.0�= -74 , = -4.0

1.0

�-Helix (4.416) Hypothetical

+4.3 1.1�= -57.1 ,= -69.7º

2.8

Collagen-helix In fibers -3.3 2.9 1.6

Structural characteristics of �-pleated sheet(1) has two forms, anti-parallel in

which the chains run in opposite directions and parallel �-pleatedsheets in which the chains extend in the same direction.

(2) Side chain pointed alternatively to either side of the sheet with the C�-C�bonds approximately perpendicular to the sheet.

(3) Mixed parallel and anti-parallel sheets are possible.(4) Globular protein contains 2-15 strands with the average of 6 strands (~25Å).

On the average each strand contains 6 residues that have the length of ~20Å.(5) pronounced right-handed twist when viewing along the polypeptide chain.

These are the important structural features of globular proteins in forming the central cores of protein.

Beta StructuresLinear group observed residues per

turn n and chirality

rise per residue,d (Å)

radius of helix r (Å)

Twisted parallel or antiparallel sheet

Abundant -2.3 3.3 1.0

Planar parallel sheet Rare 2.0 3.2 1.1

Planar antiparallelsheet

Rare 2.0 3.4 0.9

�-hairpin turns(a) hairpin (b)right-handed cross-over (c) left-handed cross-over

70% of these hairpin turns are shorter than seven residues inlength and turns containing two residues form the largest single component. In this class, types I' and II' are the most common.

Beta-hairpin turns occur between two antiparallel beta-strands

For type I' turns, residue 2 is always glycine whereas for type II' turns residue 1 is always Gly

For type I' turns, residue 2 is always glycine whereas for type II' turns residue 1 is always Gly

Reverse turns or � bendsThree favorable conformations of three consecutive peptide units (C�ito C�

i+3) with hydrogen bond between Oi and Ni+3.(a) Reverse turn I: deform 310-Helix(b) Reverse turn II: 180º flip of the peptide unit linkage residues 2 and 3 of Reverse turn I.(A bit more sterichindrance than type I turn). The (i+2) residue of type II reverse turns is nearly always glycine(c) Reverse turn III: piece of 310-Helix.

� LoopLoops are made of 6 to 16 residues that are not components of helices or � sheet and whose end-to-end are < 10Å. � loops are regularly located on the protein surface.

(illustration of all protein secondary structures)

Undefined structure: disorderOccurs at extended, charged surface groups, eg Lys, Arg, etc., N-or C-termini of polypeptide chains.

What forces determine the structure?• Primary structure - determined by covalent bonds• Secondary, Tertiary, Quaternary structures - all determined by weak forces

The forces contribute to protein stability are(A) electrostatic forcesEvaluation is based on the Columb’s law- calculate the energy of association, U, of two electric charges q1, and q2K=9.0x109 J.m.C-2

D: dielectric constant of the medium. For the interior of a protein, it is usually 3-5.(a) ionic interactionThe association of two ionic protein groups of opposite charge is known as ion pair orsalt bridge. A typical ion pair is the carboxyl group of Glu and the ammonium group of Lys. However, they are rarely seen to be buried (solveated).Ion pairs contribute little stability toward a protein’s native structure.(b) dipole-dipole interaction

van der Waals forces.van der Waals forces Arises from electrostatic interaction among permanent and/or induced dipoles.

Dipole-dipole interactions(b) –cont’d(i) Permanent dipoles interactions are important structural determinants in proteinsThese energy vary with r-3.(ii) A permanent induces a dipole moment on a neighboring group. Such dipole-induced dipole interactions are generally weaker.(iii) Nonpolar molecules have a small dipole moment resulting from the rapid fluctuation motions of their electrons. Theses so-called London-dispersion forces are extremely weak. Their association energy is proportional to r-6. The London dispersion forces play major role in determining protein’s conformation because of the great numbers of interatomic contacts in the closely packed interiors of proteins.

Hydrogen bonding forcesHydrogen bonding forces are permanently electrostatic interactions between(a) Weakly acidic group (D-H) and acceptor (A) that bears a lone pair of electrons.

(D and A can be N, O and S atom or D-H…� hydrogen bonds). The D…A distance is in the range of 2.7 to 3.1 Å. (D…A < 3.7 Å ( D-H (~1 Å HA van der Waals contact (~2.7 Å) )

(b) The association energy of H-bond is between those of covalent and van derWaals forces.

(c) covalent bond > H-bonds > van der Waals(d) H-bonds tend to be linear, but sometimes with deviated angle no less than 100º.(e) Despite the low stability of H-bonds, they provide a structural basis for protein’s

native folding pattern. (� helices and � sheet efficiently satisfies the polypeptide backbone’s H bonding requirements.)

(f) Most hydrogen bonds in proteins involved donors and acceptors that are close together in sequence. 68% of the H-bonds in proteins are between backbone atoms. Over half of the H-bonds between side chains are between charged residues.

Hydrophobic forcesHydrophobic effect causes nonpolar substances to minimize their contacts with water and amphipathicmolecules. ( detergent micells)In 1958, Walter Kauzmann pointed out that hydrophobic forces are a a major influence in causing proteins to fold into their native conformations.

micell nonpolar side chains

(d) Disulfide bondsDisulfide binds function to stabilize the three dimensional structure of protein. The reducing environment of the cytoplasm tend to diminish the stability of disulfide bonds. Therefore, almost all proteins with disulfide bonds are secreted to more oxidized extracellular environment.

Protein denaturationConformational sensitive properties, :CD, viscosity, UV, can detect protein’s folding state. Proteins unfold in a cooperative manner. At elevated temperature, any partial unfolding of the protein structure destabilized the remaining structure and resulted to the random coil.Conditions leads to denature proteins:(1) temperature(2) pH(3) detergent(4) high conc. of water soluble organic solvent(5) saltHofmeister series of salts: the tendency to stabilize proteinsGuanidine HCL, urea tend to denature proteins

Anions: SO42- > H2PO4

- > CH3COO - > Cl - > Br - > I - > ClO4- > SCN -

Cations: NH4+, Cs+, K+, Na+ > Li+ > Mg2+ > Ca2+ > Ba2+

Chaotropic agents disrupt the hydrophobic interactionsof proteins.

chaotropic

Tertiary Structure1950 (sperm whale myoglobin) John Kendrew

2000 ( DNA, RNA )

(a) globular protein secondary structure �-helix �� sheet31% �-helix, 28% �-sheet

(b) side-chain(i) val, leu, Ile, Met, Phe(ii) Arg, His, Lys, Asp, and Glu

(iii) Ser, Thr, Gln, Tyr, and Trphydrogen bonding.

Protein properties:

packing density of protein = Van der Waals envelop / total volumeprotein : ~ 0.75close packing sphere: ~0.74oil drop: 0.6~0.70

200 domain ~25ÅDomain: structural independent unit : protease release one domain without altering its structure.

domain glyceraldehyde-3-phosphatedehydrogenase NAD+

Super-secondary structure��� ��� motif : right handed crossover

conncection between two � sheet.��� � hairpin motif: antiparallel � sheet

connected by tight reverse turns��� �� motif: two antiparallel � helices

coil-coil(4) Greek key motifGroups of motifs combine in overlapping or non-overlapping ways to form the tertiary structure of a domain, which is called fold.It is know that few protein folds are unique. Ie. The number of folding motif is limited. There are about ~1000 naturally occurring folds (~600 were found)(1) � domain : exclusively a helices(2) � domains: only � sheets

(i) � sandwich (ii) � barrelsBarrels are sub-divided into (a) up and down � barrel (b) – (c) jelly roll or swiss roll barrel

(3) ��� domains: a central parallel or mixed � sheet is flanked by � helices(4) open ��sheets����� unit: (discovered by Michael Rossmann) form dinucleotide-binding site in many enzymes(Illustration)

Structural BioinformaticsIn addition to know how sequences of proteins and DNAs related in the phylogenetictree by using the sequence alignment, an equally important aspect is to know how the structures of maromolecules are displayed and compared.(a) Protein Data bank: (b) Nucleic acid database(c) Structural classification and comparison

(i) CATH class, architecture, topology, and homologous superfamily(ii) CE: find all proteins in the PDB that can be structuraly aligned with the query sequence(iii) FSSP: find structures in PDB resemble that of the query structure(iv) SCOP: classify protein structure based on generated topology.Class(All-�, all-�, ���, ���, multi-domain)Fold (secondary structure similarity)Superfamily (show distant evolutionary relationships)Family (evolutionary relationshps based on sequence and structure)

Eg: 1MBO.pdball-�, Fold: globin-like superfamily: globin-like family: globins

(v) VAST (structural alignment)

123

Quaternary Structures:Quaternary structures depend on the arrangement of two or more polypeptide chains. The primary, secondary, and tertiary structures all refer to single polypeptides. However, proteins with quaternary structure are called multimeric proteins(many subunits).

Subunit interactionMultiunit protein polypeptide chains. hemoglobinprotien subunit �2�2. subunit oligomers

subunit protomers .“ ”

quaternary structureC2, C3, C5, D2, D4, D3, T, O, I.

Symmetry:

Symmetry: (con’t)

: piconavirus 20532

Example of glutamine synthase homo-oligomer with D6 synnetry

X-ray structure reveals molecular details of this molecule.

Summary of protein structuresI. Homologous protein:

Same 3-D shapeHighly similar A.A. sequenceSame functioneg. Cytochrom C

II. Similar function-different sequenceOne domain same shapeOne domain differenteg. Dehydrogenase

III. Similar shape-different function and different sequenceSame 3-D shapeNot the same functioneg. �-barrel proteins