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

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

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

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”

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 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.

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: http://www.tops.leeds.ac.uk/ 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

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

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

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

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” 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

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

“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 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

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 http://sts-fw.bioengr.uic.edu/castp/index.php 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 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.

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 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

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

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 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

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.

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.