1. Basic protein structure and stability V: Even more protein anatomy
Biochem 565, Fall 2008
09/05/08
Cordes

2. Tertiary structure in proteins
Tertiary is the level of protein structural hierarchy above secondary, and involves:
The number and order of secondary structures in the sequence (connectivity) and their arrangement in space. This defines a protein’s tertiary fold (more on this later) also called its global topology
Pattern of contacts between side chains/backbone, including and especially contacts between residues in different regions of the sequence (long-range)
Outer surface and interior--what’s inside/outside
Limited to interactions within a single polypeptide chain--interaction between chains is quaternary

3. Supersecondary structures/structural motifs
just as there are certain secondary structure elements that are common, there are also particular arrangements of multiple secondary structure elements that are common
note that I don’t consider a beta-sheet a secondary structure element, because it is not regular and contiguous in the sequence--I consider the individual strands to be secondary structure elements, while the sheets formed from these strands are supersecondary structures (really an aspect of tertiary structure)
supersecondary structures emphasize issue of topology in protein structure
greek key motif
b-a-b motif

4. Topology: differences in connectivity
example: a four-stranded antiparallel b sheet can have many different topologies based on the order in which the four b strands are connected:
“up-and-down”
“greek key”

5. Topology: differences in handedness
example: An extremely common supersecondary structure in proteins is the beta-alpha-beta motif, in which two adjacent beta-strands are arranged in parallel and are separated in the sequence by a helix which packs against them.
if the two parallel strands are oriented to face toward you, the helix can be either above or below the plane of the strands.
huge preference for right-handed
arrangement in proteins

6. Topology/geometry: beta-sheet twisting
Beta-sheets are not flat:
they have a right or left
handed “twist”, and
essentially all beta-sheets
in proteins have a right-handed
twist like that seen in
flavodoxin (1czn) at right.
See Richardson article for naming
convention, and ideas about the
origin of a preference for the
right-handed twist.

7. Visualizing topology--TOPS cartoons
Cu/Zn superoxide dismutase
all anti-parallel
beta structure
sheet 1
sheet 2
this is a TOPS
cartoon of the
structure at left
URL for TOPS website:
But the site currently appears to be nonfunctional (under maintenance)
lines = loops
if loop enters from top, line drawn to center
if loop enters from bottom, line drawn to boundary
up triangles = up-facing b strands
down triangles = down-facing b strands
horizontal rows of triangles = b sheets (beta barrel would be a ring of triangles)
circles = helices

8. Contact maps of protein structures
-both axes are the sequence of the protein
map of Ca-Ca distances < 6 Å
In the Richardson article these are called “diagonal plots”.
near diagonal: local contacts in the sequence
off-diagonal: long-range (nonlocal) contacts
rainbow ribbon diagram
blue to red: N to C
1avg--structure of triabin

9. Contact maps of protein structures
-both axes are the sequence of the protein
map of Ca-Ca distances < 6 Å
In the Richardson article these are called “diagonal plots”.
rainbow ribbon diagram
blue to red: N to C
Structure of n15 Cro

10. Contact maps of protein structures
-both axes are the sequence of the protein
map of all heavy atom distances < 6 Å (includes side chains)
rainbow ribbon diagram
blue to red: N to C
Structure of n15 Cro

11. Surface and interior of globular proteins
solvent accessible surface
molecular surface
residue fractional accessibility
pockets and cavities
“hydrophobic core”
ordered waters in protein structures

12. “Accessible Surface” mathematically roll a sphere all around that
represent atoms as spheres w/appropriate
radii and eliminate overlapping parts...
the sphere’s
center traces
out a surface
as it rolls...
Lee & Richards, 1971
Shrake & Rupley, 1973

13. Now look at a cross-section (slice) of a protein structure:
Inner surfaces here are van der Waals. Outer surface is that traced out by the center of the sphere as it rolls around the van der Waals’ surface. If any part of the arc around a given atom is traced out, that atom is accessible to solvent. The solvent accessible surface of the atom is defined as the sum the arcs traced around an atom.
there’s not much solvent accessible surface
in the middle
van der Waals
surface
solvent
accessible
surface
from
Lee &
Richards,
1971
arc traced around atom

14. “Accessible surface”/“Molecular surface”
note: these are alternative ways of representing the same reality:
the surface which is essentially in contact with solvent

15. molecular and accessible surfaces are both useful representations, but molecular surface is more closely related to the actual atomic surfaces. This makes it somewhat better for visualizing the texture of the outer surface, as well as for assessing the shape and volume of any internal cavities.
you will hear the term Connolly surface used often, after Michael Connolly. A Connolly surface is a particular way of calculating the molecular surface. The accessible surface is also occasionally called the Richards surface, after Fred Richards.

16. Molecular surface of proteins
depiction of the corresponding
“molecular surface”--volume contained
by this surface is vdW volume plus
“interstitial volume”--spaces in between
depiction of heavy atoms (O, N,C, S) in a protein as van der Waals spheres

17. The irregular surface of proteins: pockets and cavities
a pocket is an empty concavity on a protein surface which is accessible to solvent from the outside.
a cavity or void in a protein is a pocket which has no opening to the outside. It is an interior empty space inside the protein.
Interesting website for calculation/evaluation of cavities: CASTP
Pockets and cavities can be critical features of proteins in terms of their binding behavior, and identifying them is usually a first step in structure-based ligand design etc.

18. Fractional accessibility
calculate total solvent accessible surface of protein structure (also can calculate solvent accessible surface for individual residues/sidechains within the protein)
can also model the accessible surface area in a disordered or unfolded protein using accessible surface area calculations on model tripeptides such as Ala-X-Ala or Gly-X-Gly.
from these we can calculate what fraction of the surface is buried (inaccessible to solvent) by virtue of being within the folded, native structure of the protein.
this is done by dividing the accessible surface area in the native protein structure by the accessible surface in the modelled unfolded protein. That’s the fractional accessibility. The residue fractional accessibility and side chain fractional accessibility refer to the same thing calculated for individual residues/sidechains within the structure.

19. Accessible surface area in globular protein structures
Accessible surface area As in native states of proteins is a non-linear function of molecular weight (Miller, Janin, Lesk & Chothia, 1987):
As = 6.3Mr0.73
` where Mr is molecular wt
This is an empirical
correlation but it comes
close to the expected
two-thirds power law
relating surface area to
volume or mass for a set
of bodies of similar shape
and density.

20. How much surface area is buried when a protein adopts its native structure in solution?
estimate total accessible surface area in extended/disorded polypeptide chain using the accessible surface areas in Gly-X-Gly or Ala-X-Ala models. This is a linear function of molecular weight
At = 1.48Mr + 21
the total fractional accessibility is As/At ,and the fraction of surface area buried is 1- As /At
What is the total fractional surface area buried for a protein of molecular weight 10,000? 20,000? Is the fraction higher for small proteins or large?

21. Distribution of residue fractional accessibilities
note that a sizeable group are completely buried
(hatched) or nearly completely buried
note broad distribution among non-buried residues, and mean fractional accessibility for non-buried residues
of around 0.5
note that few residues are
completely exposed to
solvent, but that fractional
accessibility of >1 is possible
from Miller et al,
1987

22. Buried residues in proteins
the fraction of buried residues (defined by 0% or 5% ASA cutoffs) increases as a function of molecular weight--for your average protein around 25% of the residues will be buried. These form the core.
size class mean Mr fraction of buried residues
0% ASA 5% ASA
small
medium
large XL
all

23. Residue fractional accessibility correlates with free energies of transfer for amino acids between water and organic solvents
(Miller, Janin, Lesk & Chothia, 1987)
(Fauchere & Pliska, 1983)
the interior of a protein is akin to a
nonpolar solvent in which the nonpolar
sidechains are buried. Polar sidechains,
on the other hand, are usually on the
surface. However, some polar side chains
do get buried, and it must also be
remembered that the backbone for every
residue is polar, including those with
nonpolar side chains. So a lot of polar
moieties do get buried in proteins.

24. The hydrophobic core of a small protein: N15 Cro
0% ASA: Pro 3 Leu 6 Ala 16 Val 27 Ile 36 Ile 44 < 5 % ASA: Met 1 Ala 17 Val 20 Gln 41 Ser 54
note that some polar residues
are buried
11 of 66 ordered residues have less than 5% ASA

25. The outer surface: water in protein structures
Structures of water-soluble proteins determined at reasonably high resolution will be decorated on their outer surfaces with water molecules (cyan balls) with relatively well-defined positions, and waters may also occur internally
Water is not just surrounding the protein--it is interacting with it
Refer back also to the section on water in the Richardson review.

26. Water interacts with protein surfaces
Most waters visible in crystal structures make hydrogen bonds to each other and/or to the protein, as donor/acceptor/both
second shell water:
only contacts other waters
first shell waters:
in contact with/
hydrogen bound
to protein
Refer back also to the section on water in the Richardson review.