Regular secondary structure:-helix, -sheet

repeating values

-helixPauling 1951-only peptide helix with in fully allowed regions-right-handed- 3.6 residues per turnpitch = 5.4 Å per turn

typically ~12 residues in protein (over 3 turns and 18 Å)

C=O (residue 1) H-bonded to N-H (residue 4)sidechains sticking out from helix

core tightly packed (atoms in vdW contact)

-sheetalso uses full H-bonding capacity of backbone (between neighboring chains)

anti-parallel or parallel orientation

not exactly completely extended (would put sidechains pointing at neighboring polypeptide and give a steric clash)

Observed =-90 - -180observed = 150-180-pleated or rippled sheet pattern-overy other sidechain points up -better geometry for C=O…H-N hydrogen bond

sheets 2-22 strands, ave.=6, up to 15 residues (small also observed)

right-handed twist (compromise between L-amino acid centers and maximizing interchain H-bonding)

Collagen, a triple helix-most abundant vertebrate protein!!!strong, stress-bearing fibers components of bone, teethcartilage, tendon, and fibrous matrices of skin and blood vessels

3 polypeptide chains, distinctive a.a. composition:33% gly15-30% pro, hyp

4-hydroxyprolyl residue3-hydroxyprolyl and 5-hydroxylysyl also occur smaller amounts

Created by modification of synthesized polypeptide chains Pro Hyp prolyl hydroxylase (requires ascorbic acid for its activity)Scurvey…..limeys…..

2° structure? Proline!!!!!

Collagen has G-X-Y repeating sequenceX-often pro, Y-often Hyp

Proline cannot form -helices (=-60°, restricted, and no N-H for H-bond to C=O)

So, collagen forms gentle left-handed helix with 3 residues per turn

And, 3 helices reverse the twist to form aright-handed 3-helical coil

Every 3rd residue through center of triple helix (so crowded that only room forgly sidechain…explainsabsolute requirement for gly every 3rd residue)

3 chains staggered, so only gly in center of coil

Each gly N-H forms strongH-bond with Pro C=O onneighboring chain to stabilize overall structure

Pro, Hyp bulky & inflexible,making assembly rigid

Collagen’s strength comesfrom: well-packed, rigid, triple helix (can’t unwind)

Crosslinking: lysyl oxidase, then: 2X allysine, aldol, His adds, 5-hydroxylysine, increases with age…tough old meat…

Non-repetitive protein structureMostly globular…several types of regular secondary struc (,,turns), irregular and unique also possible (non-repetitive , values)

Don’t confuse with random coil (what I have been calling spaghetti in class)

#2 often Pro (-60°)

#3 often Gly(no R group)

Clash!

• Random coil:disordered and rapidly fluctuating conformation assumed by denatured (fully

unfolded) proteins (imagine hydrogen bond acceptors and donors making all H-bonds with water, hydrophobics randomly burying surface with self and non-self polypeptide chains).

• Irregular Structures:in native proteins, irregular structures are no less ordered than helices or -

sheets, simply more difficult to describe

• Distortions:a.a. sequence variations, overall structure of folded protein can distort regular

conformations of secondary structural elements. -1st and last turns of helix (don’t have all H-bonds)-helix capping (Gln/Asn sidechain folds back to H-bond with backbone exposed

C=O groups…)--bulge (1 residue in -strand not H-bonded…pokes out)-Pro kinks helices & sheets-(ii+3,4 steric clashes big sidechains in a helix)

Propensities of a.a. in known structures are useful to predict 2° structure (Pro,Gly in turns, between alpha/beta structures)

Protein structure determinationProtein Data Bank (www.rcsb.org), ~30,000 structures, downloadable coordinate files

X-ray crystallography (direct imaging of molecules)From optical principles, error associated with locating an object is on the order of the wavelength of light used to observe it (covalent bonds and X-rays both ~1.5 Å)visible light ~4000 Å, too long…

Crystals diffract X-rays onto a radiation counter (or photographic film…)

X-rays now normally produced from a particle accelerator, called a synchrotron (Grenoble, Los Alamos, huge national user facilities…).

Intensities (darkness of spots) used to calculate mathematically the position of electrons that diffracted the X-ray.

X-rays interact primarily with electrons, not nuclei. Therefore, crystallographers plot electron density maps and then try to model protein residues into the shape of the map.

Every ( e.g. Tyrosine 56 beta carbon) atom in the lattice of protein molecules deflects the incident X-ray with the same angle… You don’t have to know how diffraction works, only that we can reconstruct electron density from the diffraction pattern.

X-ray, cont’d.1Even though structures presented as atomic models, most structures are less than atomic resolution!!!!!

-Proteins arranged as repeating, 3-dimensional lattices-Protein crystals are 40-60% water(must be in aqueous conditions for their structural integrity…therefore proteincrystals are soft, jellylike in consistency)-Molecules in crystal are typically disordered by 1 Å or more-Resolution is typically 1.5-3.0Å, but some more ordered (better resolution) and some worse…

C-C bond length ~1.5 Å

Model hydrogen positions andN/O identity based on nearby groupsCan lead to bias towards ideal,“normal” geometry and H-bonding

Accuracy and feasibility of crystal structure analysis depends on resolution -Trace distinctive backbone, deduce orientations of sidechains, but Ile, Leu, Thr and Val hard to distinguish.-Hydrogen only observed at less than 1.2 Å, and N and O are hard to distinguish*Utilize primary sequence to fit it into the density

clearly shows atoms

(1b)

X-ray, cont’d.2Crystalline proteins mostly in native conformation

•Protein basically solvated by crystallization solution except where it contactsneighboring protein molecules (small patches called crystal contacts) (40-60% water is similar to a living cell)

•Proteins can be crystallized in more than one crystal form, and still give the same structure…your book claims that NMR vs. crystal comparison shows proteins with same structure typically (BUT, there can be important differences!)

•Many enzymes are catalytically active in the crystalline state, and since they must have their catalytic sidechains perfectly oriented, this is strong evidence of its occurrence in the crystal

Disordered portions of proteins (loops, termini, unfolded elements) do not diffract X-rays, because the position of the atoms in each molecule in the crystal is not the same. Therefore, these portions in an otherwise ordered crystal give no structural information and are left out of structure models.