Basic protein structure and stability V:

Even more protein anatomy

Biochem 565, Fall 2008

09/05/08

Cordes

Tertiary structure in proteinsTertiary 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

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

motif greek key motif

Topology: differences in connectivity

“greek key”“up-and-down”

• example: a four-stranded antiparallel sheet can have many different topologies based on the order in which the four strands are connected:

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

Topology/geometry: beta-sheet twisting

Beta-sheets are not flat:they have a right or lefthanded “twist”, and essentially all beta-sheetsin proteins have a right-handedtwist like that seen in flavodoxin (1czn) at right.

See Richardson article for naming convention, and ideas about theorigin of a preference for theright-handed twist.

Visualizing topology--TOPS cartoons

• lines = loops• if loop enters from top, line drawn to center• if loop enters from bottom, line drawn to boundary

• up triangles = up-facing strands• down triangles = down-facing strands• horizontal rows of triangles = sheets (beta barrel would be a ring of triangles)• circles = helices

all anti-parallelbeta structure

Cu/Zn superoxide dismutase

this is a TOPScartoon of thestructure at left

sheet 1

sheet 2

Contact maps of protein structures

1avg--structure of triabin

map of C-C distances < 6 Å

near diagonal: local contacts in the sequence

off-diagonal: long-range (nonlocal) contacts

rainbow ribbon diagramblue to red: N to C

-both axes are the sequence of the protein

Contact maps of protein structures

Structure of n15 Cro

-both axes are the sequence of the protein

rainbow ribbon diagramblue to red: N to C

map of C-C distances < 6 Å

Contact maps of protein structures

Structure of n15 Cro

-both axes are the sequence of the protein

rainbow ribbon diagramblue to red: N to C

map of all heavy atom distances < 6 Å (includes side chains)

Surface and interior of globular proteins

solvent accessible surface

molecular surface

residue fractional accessibility

pockets and cavities

“hydrophobic core”

ordered waters in protein structures

“Accessible Surface”

Lee & Richards, 1971Shrake & Rupley, 1973

represent atoms as spheres w/appropriateradii and eliminate overlapping parts...

mathematically roll asphere all around thatsurface...

the sphere’scenter tracesout a surfaceas it rolls...

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.

solventaccessiblesurface from

Lee &Richards,1971

van der Waalssurface

arc traced around atom

there’s not much solvent accessible surface in the middle

“Accessible surface”/“Molecular surface”

note: these are alternative ways of representing the same reality:the surface which is essentially in contact with solvent

• 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.

Molecular surface of proteins

depiction of heavy atoms (O, N,C, S) in a protein as van der Waals spheres

depiction of the corresponding “molecular surface”--volume containedby this surface is vdW volume plus“interstitial volume”--spaces in between

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.

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.

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.

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 empiricalcorrelation but it comesclose to the expectedtwo-thirds power law relating surface area tovolume or mass for a setof bodies of similar shapeand density.

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?

Distribution of residue fractional accessibilities

note broad distribution among non-buried residues, and mean fractional accessibility for non-buried residuesof around 0.5

note that few residues arecompletely exposed to solvent, but that fractionalaccessibility of >1 is possible

from Miller et al,1987

note that a sizeable group are completely buried(hatched) or nearly completely buried

Buried residues in proteins

size class mean Mr fraction of buried residues0% ASA 5% ASA

small 8000 0.070 0.154medium 16000 0.107 0.240large 25000 0.139 0.309XL 34000 0.155 0.324all 0.118 0.257

•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.

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.

The hydrophobic core of a small protein: N15 Cro

0% ASA:Pro 3Leu 6Ala 16Val 27Ile 36Ile 44< 5 % ASA:Met 1Ala 17Val 20Gln 41Ser 54

11 of 66 ordered residues have less than 5% ASA

note that some polar residuesare buried

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

Water interacts with protein surfaces

second shell water:only contacts other waters

first shell waters:in contact with/hydrogen boundto protein

Most waters visible in crystal structures make hydrogen bonds to each other and/or to the protein, as donor/acceptor/both