EFB325 Cell Physiology

Proteins: structure/folding/ligand binding

Rotation around the peptide bond is restricted, but rotation around the alpha-carbon can occur

• results in nearly infinite combinations of bends and twists of a polypeptide chain

For a protein to function correctly, it must fold/twist/pack/bend into the one proper shape

This shape is primarily determined by the sequence of amino acids and their chemical properties

Chemical interactions involved in protein structure

• hydrophobic interactions (hydrophobic residues go to the middle of a protein)
• hydrogen bonds (can involve atoms in the peptide bond or the side groups)
• ionic bonds (usually atoms in charged R groups of the amino acids)
• disulfide bridges (covalent bonds between the side groups of two cysteine residues)

                -C-SH + HS-C- -----> -C-S-S-C-

• van der Waals attractions (determine how close atoms get to one another)

Levels of structural organization (not only in proteins)

Primary structure

• simply the sequence of residues (amino acids or nucleotides)
• genetically determined
• all the higher levels of structure ultimately depend on the primary sequence

Secondary structure

= local interactions between adjacent amino acids in a polypeptide leading to folding into particular types of 3-D structures

• alpha helix =is shaped like a spring in a portion of a polypeptide
• every amino acid has a hydrogen bond to the fourth amino acid in sequence (3.6 aa's per turn of the helix)
• involving the atoms in the peptide bonds
• R groups stick out from the cylinder
  
• beta pleated sheet=parallel or anti-parallel polypeptide chains
• hydrogen bonds between atoms in the peptide bonds
• forms a sheet w/R groups pointing alternately up then down

Tertiary structure

• folding, coiling, twisting into the most stable conformation of the entire polypeptide
• secondary structure elements as well as random coil
• can involve covalent bonds between Cys residues upon oxidation=disulfide bridges
  
• proteins structures can be:
• fibrous=simple, elongated structure, often filamentous (like keratin - forms a coiled-coil, collagen forms a triple helix structure)
• globular=polypeptide folds into compact shape like a ball with irregular surface

Quaternary structure

• organization, folding, and packing of multiple polypeptides together (subunits of dimers, tetramers, multimers)
• same type of bonds involved as in tertiary structure

The information needed to fold and assemble into the proper conformation is for the most part inherent to the primary sequence

• high degree of self-assembly
• must occur in proper oxidizing environment (to form disulfide bonds)

There are many, many possible final 3-D conformations, but usually only one is correct for the proper structure/function of the protein

• the folding of a protein must avoid those incorrect conformations

Many proteins represent a combination of different regions=domains

• tend to fold independently of other regions of the protein
• each tends to perform a certain function, such as . . .region of interaction with another protein or catalytic region of an enzyme
• domains tend to be encoded in a single exon, which has allowed domain shuffling' and gene recombination to generate 'new' types of proteins over millions of years
• one protein may perform more than one catalytic activity

What are conditions that disrupt this folding and assembly?

(Things that disrupt hydrogen, ionic, and disulfide bonds, hydrophobic interactions)

• heat
• salt (ionic compounds)
• oxygen (oxidizing environment)
• detergent
• pH

When protein structure is disrupted, the protein is denatured

Under acute heat stress (heat shock) essentially all organisms stop making most of their regular proteins and instead produce a special set of proteins

• heat shock proteins=HSPs
• some of the HSPs act as molecular chaperones, in order to maintain proper protein structure in high temperature

Folding is often aided by molecular chaperones

• protein that binds to newly synthesized polypeptides, but is released from the final structure
• more common with proteins that are large or have many subunits (many possible paths of folding/assembly)
• prevents incorrect folding pathways, but don't provide additional information
• 20% of all proteins are assisted by hsp70 (hsp=heat shock protein; 70 Da Mol. Wt.); 10% assisted by hsp60
• they use ATP and bind to exposed hydrophobic patches on proteins

Proteins that are not properly folded are degraded by proteinases in a structure called the proteosome - this cleaves the polypeptides between each amino acid, so the amino acids can be 'reused' to make new proteins

Some neurodegenerative diseases result from the aggregation of misfolded proteins that are resistant to proteolysis

• the misfolded proteins assume a shape with a continuous stack of beta sheets, called a cross-beta filament
• protein aggregates accumulate and kill cells - they are called amyloids
• the accumulation of these amyloids can disrupt tissue function, especially in brain and nervous tissue
• these diseases include Alzheimer's disease and Huntington's disease

There are infectious diseases that also involve the formation of amyloids due to aggregation of protease-resistant misfolded proteins = types of spongiform encephalopathy

• scrapie of sheep - discovered in 1943 when an inoculum from an infected sheep transmitted the disease
• kuru in New Guinea - transmitted through ritual cannibalism
• bovine spongiform encephalopathy (BSE or mad cow disease)
• Creutzfeldt-Jacob disease - can arise spontaneously with very low frequency
• chronic wasting disease in elk and deer

The infectious agent of scrapie was purified and found to be a protein by Stanley Prusiner in 1982.

• the protein - he called prion protein (PrP) - is normally produced in animals
• the disease occurs when this protein misfolds and becomes highly resistant to proteolysis
• the misfolded protein forms aggregates with high proportion of beta sheet
• the misfolded protein can induce the misfolding of other proteins, thus it is an infectious protein agent
• Prusiner won the Nobel Prize in 1997

Ligand binding

Proteins bind other molecules (ligands) through a combination of many weak chemical interactions

• a binding site is usually formed by the juxtaposition of amino acids from different parts of a protein
• this forms a pocket/crevice/cavity where the ligand fits and will be held by the combination of ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions
• the 3-dimensional structure of the protein and the particular amino acids involved are critical in forming the binding site
• the binding site is usually very specific for the proper ligand
• different binding sites can bind ligands with varying strengths

Antibodies are proteins that can bind very specifically to ligands (=antigen)

• your body can make antibodies that can bind a nearly infinite variety of antigens by varying the amino acid sequence of the binding site
• any one antibody only binds a single type of antigen

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