PROTEINS

 

I.              Read section in chap 6 on ³Enzymes: Biological catalysts and Molecular Structure Determines Enzyme function (6th: p102-108; 7th:p113-119)

II.            Proteins (CHNOS) (made up of amino acids)

III.         Introduction - General functions

A.      Enzymes - catalyze chemical reactions. Examples: hexokinase, ATPase, amylase. Will be discussed in detail later. Enzyme biochemical chart

B.      Energy - the C-H and C-C bonds of ANY protein or ANY amino acid can be used as a source of energy. A problem with using proteins as an energy source is that one is left with nitrogen as a waste (usually in the forms of ammonia, urea, or uric acid) which is somewhat toxic and somewhat difficult to remove from the living system.

C.      Structure (collagen of bone, keratin and elastin of skin)

D.      Others:

1.      Microtubules, Actin filaments, intermediate filaments: actin, tubulin, myosin

2.      Regulatory: hormones, neurotransmitter peptides

3.      Membrane proteins: Receptors, transport, antigens.

4.      Antibodies.

IV.         The structure of proteins.

A.      Two general categories:

1.      Globular (usually soluble, usually enzymatic)

2.      Fibrous (usually insoluble, usually structural)

B.      Amino acids - the building blocks of proteins.(proteins are an amino acid polymer. When the number of amino acids is small then it is often referred to as a polypeptide).

1.      Amino acid structure (there are several ways of writing the same structure:)

 

R (residual group) is one of 20 possible organic groups.


 

 


(this is a zwitterion because it has two full charges while overall it is an uncharged molecule)

 

2.      20 (or so) amino acids depending upon the R group.

a)       Varying degrees of hydrophobicity, charges, sizes, etc.

b)       Show some examples

(1)          hydrophilic

(a)          polar

(b)          charged, polar

(2)          hydrophobic - uncharged R groups

(3)          sulfated - namely cysteine and methionine.

(4)          Cysteine can covalently bond its R group to another R group (the only amino acid with this property) by forming -S-S- bonds through an oxidation-reduction reaction:

aa-CH3-S-H  + H-S-CH3-aa à aa-CH3-S-S-CH3-aa + H2

 

(5)          ringed backbone - namely proline

3.      Isoelectric point -the pH at which the overall charge is zero. (It can be approximated as the average of the pKs)

C.      Peptide bond

 

D.      Several amino acids linked together in a chain is known as a polypeptide chain. A large polypeptide (~50 or more amino acids ) that has folded into a functional unit would be called a protein.

V.           Proteins - primary structure

A.      The primary structure is the amino acid sequence of the protein.

B.      The first primary structure determined was for insulin by Sanger (1958 Nobel Prize) (show transparency of insulin). Achieved by biochemically "clipping" the protein from the end into identifiable pieces.

1.      Two polypeptide chains (21 aa and 30 aa) linked by cysteines.

2.      The way insulin is formed was later discovered. It IS formed as a single chain, the -S-S- bonds of cysteine form, and then a mid section is cut off.

3.      Insulin from various animals varies only slightly in its primary structure(positions 8,9, and 10 of the a chain vary  most frequently; man and rabbits are weird in that they vary in the terminal aa(30) of the b chain). Despite the differences, bovine insulin can be used in man with similar effectiveness.

C.      Enzymes typically have 100 to 500 aa.

D.      Instruments now "automatically" determine primary structure, BUT now primary structure is now usually and easily determined by sequencing the DNA and then using the DNA code to determine the associated protein.

VI.         Secondary structure: precise and repeated folding due to hydrogen bonding with respect to the amino acid backbone.

A.      Pauling and Cory (Nobel Prize 1954) studied the flexibility in the covalent bonds of a peptide backbone structure as well as the partial charges of the backbone structure and determined that peptides could bend EVERY FOURTH (actually every 3.4) amino acid (alpha helix).(alpha means it twists in a right handed, clockwise manner) Another highly probable situation would be to bind one section of peptide chain to another via hydrogen bonds (beta pleated sheet)

1.      Proteins may have a high proportion of a helix or a high proportion of ß pleated sheet or any combination thereof.

a)       Show ribbon diagrams and the fact that many proteins fall in similar classes.

b)       Show that the ß sheet structures are either parallel or antiparallel and often sit askew of each other.

c)       Show that the a helix is dipolar. They are also either parallel or antiparallel and often sit askew of each other.

d)       None of these chains run too many amino acids in length.

e)       Glycine and proline are secondary structure breakers and are often found where the protein bends.

2.      Structural proteins will tend to be configured into a pleated sheet.

3.      Random coil is (more or less) the structureless area and is important in that is the area where the protein can most easily bend.

VII.      Tertiary structure - three dimensional folding due to the R groups. (about 200 have been done so far)

A.      What causes the protein to fold?

1.      Covalent bonding between cysteine groups.

2.      Hydrogen bonding between R groups.

3.      Ionic bonding between R groups

4.      Hydrophobic bonding - Van der Waals forces.

5.      Note that the environment influences how the protein will fold.

B.      First tertiary structure of a protein was fully discovered using myoglobin by Kendrew (Nobel Prize 1962) using X-ray diffraction.

C.      Most water soluble proteins have a hydrophobic interior and a hydrophilic exterior.

D.      A common way for enzymes to denature is to unfold either because of hydrogen bond breakage (often due to pH or temperature) or oxidants or reductants that unnaturally break or form disulfide bridges. In any case, the active site is affected, which in turn affects the activity.

E.      The final protein structure depends upon how the protein folds as it comes off of the ribosome and any subsequent processing (i.e. the functional folded protein may not be the way the protein would fold if it were completely unfolded and allowed to refold.)

F.       Sometimes there are chaperonins that help proteins fold correctly (after being made off of a ribosome or where there is a tendency for denaturing.) Chaperonins themselves are proteins.

G.      Prions: (pre-on) are proteins that infectious qualities, such as in mad cows disease. There is a normal animal brain protein (including humans) called PrP. This protein has about 45% a-helix and is virtually devoid of b pleated-sheet and forms into a particular tertiary structure. The disease form of this protein does not seem to be a genetic mutant (i.e. the DNA sequence is ok and the primary structure of the normal and diseased form are identical!). However the diseased form, PrPSc, does have a different secondary and tertiary structure (about 30% a-helix and 45% b pleated-sheet). This, in itself is not so surprising because we know that proteins can take on different shapes. The weird thing about this protein that this weird shape seems to induce the same weird shape in a normally structured protein when the two come in contact. Now the newly shaped one can induce others to change shape, Š and hence it acts like a spreading disease. The improperly shaped protein no longer functions properly and the lack of this function is what causes the brain to malfunction.

VIII.    Quaternary Structure = hydrogen bonding between globular polypeptide chains. Example - hemoglobin with 4 polypeptide chains (subunits) held together by hydrogen bonds. Usually the function of the total quaternary structure is 'better' than the function of the sum of the individual protein chains.

IX.         Conjugated proteins.

A.      A prosthetic group is a covalently bonded non-protein molecule bounded to a protein that in some way helps the overall function of the protein. The prosthetic group is usually :

1.      some organic molecule that involves pigmentation. (Chromoprotein). These are often derived from what we call vitamins. Examples: heme group of hemoglobin , myoglobin, and cytochromes. Rhodopsin of the eye would be another example.

2.      a sugar molecule (glycoprotein). Surface proteins on membranes often have these and are important for recognition of that cell. (Albumin, follicle stimulating hormone, peroxidase, ...)

3.      a lipid molecule (lipoproteins)

B.      Cofactors are inorganic ions that temporaily bind to an enzyme and critical to its function. For example Mg++ is critical for all ATPases to work.

C.      Coenzymes are small organic molecules that are required for the action of the enzyme. :