Lecture 25 April 2005

A wide variety of modifications exist. Carbohydrates are covalently-attached to proteins through the oxygens of the side chains of Serine or Threonine (O-linked), or the nitrogens of the side chains of Asparagine (N-linked).

Generally, glycosylation is used in extracellular or surface-attached proteins for (1) recognition, or (2) protection (Fig. 3-40).

Simple glycosylation occurs in yeast. It is much more prevalent and complex in mammals.

Glycosylation typically proceeds through addition of a core oligosaccharide, followed by trimming and further glycosylation. N-linked and O-linked glycosylation start with different cores. (Fig. 3-41)

Lipidation provides a way to target proteins to membrane surfaces, sometimes in a reversible manner, and sometimes to a specific membrane. There are 4 types of lipid modification:

Methylation of lysine and arginine residues on proteins found in the nuclei of eukaryotes is common. S-adenosinemethionine is the methyl donor in the reaction. Arginines can be mono- or di-methylated. Lysines can be mono- di- or tri-methylated. (Fig. 3-47) These modifications do not change the charge, but they add hydrophobic bulk, and eliminate H-bond donors. They typically affect protein-protein interactions among proteins involved in regulating transcription.

The N-termini of many proteins are acetylated. This is usually permanent, and provides protection from proteases. The epsilon-amino groups of lysine residues can also be acetylated, and these are usually deacetylated by other enzymes. For example, in the case of histones, histone acetyltransferases and histone deacetylases (Fig. 3-48).

SUMO is a small ubiquitin-like Modifier protein. It is covalently attached to other proteins via the ε-amino group of a lysine residue, similar to ubiquination. (Fig. 3-49). There is a consensus sequence in which the lysine is usually found: ψ-K-X-E, where the first residue is a large hydrophobic and X is any amino acid. (Jmol)

The use of NO, nitric oxide, as a protein modifier is widespread throughout living organisms. NO is a gaseous molecule that is produced by an enzyme nitric oxide synthase. It reacts with cysteine to form nitrosylated cysteine. (Fig. 4-50). NO also reacts with metal ions, replacing ligands. How these reactions are reversed is not understood.

Prions

Prion diseases such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) are neurodegenerative diseases in which the prion protein is the primary, if not the only, infectious agent.

CJD can be (1) sporadic, (2) genetic, or (3) infectious. The prion protein (PrP-c) occurs in normal cells, on the outside surface of neurons, but a conformationally-altered form called PrP-Sc (Scrapie form) is thought to be the infectious agent. Scrapie is the name for this disease originally discovered in sheep and goats. The altered form of the PrP has been shown to be more resistant to protease digestion, and to contain increased amount of β-sheet. The structure of PrP-c from several mammals has been determined by NMR. PrP-Sc forms insoluble fibers and therefore is difficult to study by NMR. A monoclonal antibody has been found that is specific for PrP-Sc. Crystal structures of truncated, globular PrP from the C-terrminus have been obtained. The N-terminus contains copper ion binding sites.

PrP: about 250 amino acids

22 aa make an N-terminal signal sequence
23 aa from the C-terrminus are cleaved after the attachment of a GPI anchor to Ser 231
a disulfide forms between Cys 179 and Cys 214
glycosylation occurs at Asn 181 and Asn 197
The N-terminus appears to be highly disordered.
The N-terminus contains 4 octa-repeats (PHGGGWGQ) and one near repeat (PQGGGTWGQ) that are thought to bind copper ions.
Tertiary structure of region 121-231 has 3 α-helices and 2 β-strands.

(Jmol) The original NMR structure of a segment of the mouse PrP
(Jmol) A domain-swapped dimeric human PrP indicates a possible mechanism of oligomerization.
(Jmol) Possible differences between PrP and its infectious form PrP-Sc

Short review:

Weissmann C
Molecular genetics of transmissible spongiform encephalopathies.
J Biol Chem 1999 Jan 1;274(1):3-6

Korth C, Stierli B, Streit P, Moser M, Schaller O, Fischer R, Schulz-Schaeffer W, Kretzschmar H, Raeber A, Braun U, Ehrensperger F, Hornemann S, Glockshuber R, Riek R, Billeter M, Wuthrich K, Oesch B
Prion (PrPSc)-specific epitope defined by a monoclonal antibody.
Nature 1997 Nov 6;390(6655):74-7

Alonso DO, DeArmond SJ, Cohen FE, Daggett V
Mapping the early steps in the pH-induced conformational conversion of the prion protein.
Proc Natl Acad Sci U S A 2001 Mar 13;98(6):2985-9

Knaus KJ, Morillas M, Swietnicki W, Malone M, Surewicz WK, Yee VC.
Crystal structure of the human prion protein reveals a mechanism for oligomerization.
Nat Struct Biol. 2001 Sep;8(9):770-4.

Copper binding

Viles JH, Cohen FE, Prusiner SB, Goodin DB, Wright PE, Dyson HJ
Copper binding to the prion protein: structural implications of four identical cooperative binding sites.
Proc Natl Acad Sci U S A 1999 Mar 2;96(5):2042-7 Full text

Whittal RM, Ball HL, Cohen FE, Burlingame AL, Prusiner SB, Baldwin MA
Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry
Protein Sci 2000 Feb;9(2):332-43

Yeast prions are an example of amyloid proteins (Fig. 4-53)

True HL, Lindquist SL
A yeast prion provides a mechanism for genetic variation and phenotypic diversity.
Nature 2000 Sep 28;407(6803):477-83

Balbirnie M, Grothe R, Eisenberg DS
An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid
Proc Natl Acad Sci U S A 2001 Feb 27;98(5):2375-2380

Serpins

Serpins are another class of proteins that can be considered metastable. Serpins are proteins that irreversibly inhibit serine proteases. For example, anti-thrombin is a serpin that inhibits thrombin, part of the coagulation pathway.

The action of a serpin was discovered in the case of trypsin. A loop in the alpha1;-antitrypsin covalently reacts with the catalytic Serine in trypsin. This aduct is essentially the acyl enzyme intermediate. Trypsin is unable to carry the reaction forward, because this first step triggers a conformational change in the antitrypsin. The loop is connverted into a beta;-strand and is inserted into a beta;-sheet of the antitrypsin. Trypsin, covalently attached, is propelled from one end of the antitrypsin to the other. Trypsin is partly unfolded, and becomes completely inactivated. It is sensitive to proteases.

This has been called "Inhibition by Deformation". (Fig. 4-54)

View of Membrane Proteins from the lab of Stephen White

Membrane proteins of known structure

Membrane proteins

It is difficult to crystallize membrane proteins, because they require detergents for solubilization. To crystallize, the detergent must be reduced without leading to non-specific aggregation. There are only 89 unique proteins in the PDB currently. The pace of new structures is exponential, but greatly lags the expansion of the overall PDB.

White SH
The progress of membrane protein structure determination.
Protein Sci. 2004 Jul;13(7):1948-9.

Figure

The typical membrane is 40-60Å across. It includes a central, nonpolar region of 30Å filled by fatty acyl chains of the phospholipids, and two more polar regions at each surface. (Fig. 1-29)

Most membrane proteins have transmembrane domains of bundles of alpha;-helices (Fig. 1-30) or antiparallel beta;-barrels (Fig. 1-32). (Jmol-8 strands) (Jmol-12 strands)

There are also membrane proteins, called monotopic, which are held to one surface of a membrane by helices that enter only one leaflet of the bilayer. (Jmol)

By sequence analysis it is fairly easy to detect the regions of 20 consecutive amino acids of nonpolar character , which are necessary, in general, to span a lipid bilayer (Fig. 1-31). PredictProtein (use HTM) and other servers can also be used to do this. It is more difficult to predict the transmembrane regions of β-sheet membrane proteins.

In addition to the nonpolar amino acids that populate the transmembrane spans: ALIVMF, other residues can be found also:

NSTC are fairly common, as they can make H-bonds to the backbones of other helices.
P is also fairly common in membrane helices, for reasons that are not yet clear.
G is found at close contact sites between helices.
WY are often found at the lipid-aqueous interface, due to the amphipathic nature of their side chains.

This protein is found in the "purple membranes" of halophilic bacteria. It has 248 amino acids and it was known from early EM work by Richard Henderson that it has seven α-helical transmembrane spans. It functions as a light-driven proton pump. It pumps protons out, so that it builds a conventional proton gradient that can be used for ATP synthesis, or coupled ion transport. It absorbs light through a retinal group that is covalently attached to Lys 216. (Jmol)

Water, glycerol and ammonia all can be transported across biological membranes by members of a family of transport proteins. These transporters appear in all branches of life. Humans have at least 10 such genes. They can be recognized by a signature sequence, and an overall duplication. A curious feature is that the two, related halves of the protein have opposite orientation in the membrane. A second unusual feature in the case of aquaporins is that a conserved NPA sequence is part of a half-spanning segment. A loop extends halfway through the bilayer and then returns to the original surface as an α-helix. The 2 asparagines interact at the middle of the membrane. This helps to form the very tight pathway for the particular molecule that is transported. Furthermore, these transporters form tetramers of C4 symmetry, and the central channel is an ion channel, in some cases. (Jmol of a glycerolporin)