Учебно-методический комплекс дисциплины " Basis of biochemistry " Для специальности

Modul 3. Proteins.

Primary structure of proteins. Secondary, tertiary and quaternary structures. Chemical properties and methods of definition of primary structure of proteins. Classification of proteins. The role of proteins in a food.


Peptides and Protein Primary Structure

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  Peptide bond formation: Note that a peptide bond is simply an amide bond between the alpha carboxyl and amino groups of amino acids. If we write the reacting groups in their unionized (acid and amine) forms, then we can see the reaction takes place with the loss of the elements of water, via an attack of the lone-pair electrons of the amine on the carbonyl carbon of the carboxyl group:
  

 

Now that we have looked at peptide bond formation, we next want to look at the structure of this bond and the sequence of amino acid residues (primary structures) of proteins. (Note that "residue" refers to the remainder of a molecule after it is incorporated into a polymer.)

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  The peptide bond is formed with the elimination of water, giving a planar bond between the carboxyl carbon and the amino nitrogen. [overhead 5.8 MvH] This is due to the partial double bond character on the amide/peptide bond as seen in the shorter bond length (0.133 nm vs. 0.146 nm). [overhead 7-2, V&V] This bond is nearly always trans in proteins due to steric interactions of the amide hydrogen and oxygen, except for proline.
  
• 
  Linear peptides will have free amino- and carboxy- terminal groups. Thus they will exhibit titration curves similar to a free amino acid, but with the pK_a values shifted closer to simple acid and amine values (there will be no charge stabilization).
  
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  By convention the amino terminal residue is written on the left progressing to the carboxyl terminal residue on the right: ⁺H₃N-aa-aa-aa-aa-CO₂^-.
  
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  Can determine the composition of a peptide by acid hydrolysis and amino acid analysis.
  
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  Can sequence proteins by specific enzyme and chemical hydrolysis to give peptides which can then be run through sequenators (up to about 100 aa's).
  
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  Amino acid sequences have been used to help determine relatedness of organisms.
  

 

3-D Structure of Proteins

Overview: Proteins are commonly large (MW > 6,000), globular molecules serving many functions.

Proteins are complex systems - difficult to understand at a fundamental structural level. Thus we search for patterns using normal perceptual tools: regularity, clustering, cleavage/separation/emptiness.

We are then able to discern alpha helices, beta sheets, beta turns, and "random" regions. 3₁₀ helical regions show up with computer searches. None of these is necessarily more or less random than others, they are simply easier or more difficult for us to perceive as ordered. They exist through our rationalization. Often structural elements also appear to serve a functional role, thou this is through our dissection of the molecular machine.

Look at theoretical possibilities resulting from the available bond angles around the peptide bond system

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  Most peptide bonds are trans because of reduced steric hindrance. Most exceptions are with proline which has nearly equal hindrance in both cis and trans [overhead 5.8 P]
  
• 
  Any rotation in the peptide chain will therefore take place around the two bonds of the alpha carbon, referred to as the phi (f) and psi (y) bonds. There are a restricted number of angles which these bonds can achieve (Figure 4.8) [overhead 5.9 P, V&V 7.6]. Of course the range of angles will be further reduced due to side chains.
  
• 
  If we assume hard spherical atoms with van der Waals radii, we can determine the accessible phi (f) and psi (y) angles. This procedure was followed by Ramachandran to produce the Ramachandran plot, an example is seen in Figure 4.9 of your text [overhead 6.2, MvH; 7.7 V&V].
  
  • 
    There are only a few regions of possible angles available to the alpha carbon bonds as shown on this plot.
    
  • 
    Note that the common secondary structures, the alpha helix, the beta strand, and the collagen triple helix all occur in these regions.
    
  • 
    Of course real atoms are somewhat compressible and real bonds can bend a little, so we might wonder how this plot stacks up to reality. A study of the distribution of conformation angles of a thousand amino acid residues in eight proteins as determined by x-ray diffraction showed that most of the values do indeed fall in the predicted regions. Most of the residues outside of these regions are glycines, with the least restriction.
    

Let's go back and look at overall shape and interpret it. Look for substructures that recur in various molecules. Perhaps we see a globule is made of subglobules. Look closer and we see alpha helices and beta structures. Finally we can discern aa residues.

In order to understand and categorize their organization, protein structure has been divided into four hierarchical levels and a couple of sublevels:

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  Primary structure (1°) : the linear order or sequence of peptide bonded amino acid residues, beginning at the N-terminus. (Characteristic bond type: covalent.)
  
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  Secondary structure (2°): the steric relations of residues nearby in the primary structure which give rise to local regularities of conformation. These structures are maintained by hydrogen bonds between peptide bond carbonyl oxygens and amide hydrogens. The major secondary structural elements are the alpha helix and the beta strand. (Characteristic bond type: hydrogen.)
  

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  Tertiary structure (3°): the steric relations of residues distant in the primary sequence; the overall folding pattern of a single covalently linked molecule. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals, disulfide.)
  
  • 
    Super secondary structure (motifs): defined associations of secondary structural elements. (Characteristic bond type: hydrogen & hydrophobic.)
    
  • 
    Domains: independent folding regions within a protein. The group/pattern of secondary structures forming a Domain's tertiary structure is called a Fold. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals.)
    
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  Quarternary structure (4°): the association of two or more independent proteins via non-covalent forces to give a multimeric protein. The individual peptide units of this protein are referred to as subunits, and they may be identical or different from one another. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals.)  
  

3-D Structure of Proteins 2

Secondary Structure

Tertiary structure (3°): the steric relations of residues distant in the primary sequence; the overall folding pattern of a single covalently linked molecule. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals, disulfide.)

• 
  Super secondary structure (motifs): defined associations of secondary structural elements. (Characteristic bond type: hydrogen & hydrophobic.)
  
• 
  Domains: independent folding regions within a protein. The group/pattern of secondary structures forming a Domain's tertiary structure is called a Fold. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals.)
  

 

Last time looked at what is possible given the bond angles etc. between amino acid residues. Now can look at specific structures.

Alpha helix: (Figure 4.10, pg 90 of your text) [overhead 2.31 S, 5.15 P] The most frequent secondary structure is the right-handed a-helix.

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  In this cylinder-like structure the amino acid residues curl around in a spring/rod-like structure.
  
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  There is a rise/residue (movement along the axis) of 0.15 nm and a pitch (rise/turn) of 0.54 nm.
  
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  There are 3.6 residues per turn and 13 atoms/H-bonded "ring" - this makes it a 3.6₁₃ helix.
  
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  Very importantly, the H-bonds are nearly linear and therefore of near maximum strength. The side chains of the helix stick out from the sides.
  
  • 
    The stability of the helix is determined in part by the side chains. Thus glycine allows too much rotational freedom to favor this structure, while very large or like charged side chains can also destabilize it.
    
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  As you might expect a proline residue stops a helix abruptly since proline' s angles are not accommodated in the helix.
  

Beta Strand: (Figure 4.15, pg 93 of your text) [overhead 5.19 P] The next secondary structural element is the beta-strand, which is seen in the supersecondary structures called parallel and anti-parallel beta sheets [overheads 7.16 & 17 V&V].

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  The beta strand is in a sense an abstract structure, since, unlike the a-helix, a beta-strand does not exist alone, there is always another strand to make a sheet.
  
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  In the older literature beta-sheets are considered secondary structures, but they are more consistently considered super secondary with the current nomenclature.
  
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  Beta strands are nearly fully extended, thus they have very little extensibility (stretch).
  
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  Beta strands are stabilized by hydrogen bonding to adjacent beta-strands. Thus they are stabilized by inter-strand H-bonds whereas a-helices are stabilized by intra-strand H-bonds.
  

Aside: Fibrous proteins: alpha-keratin (hair etc., alpha-helix based) [overhead 7-11 V&V, 7-25 & 26]; stretched alpha-keratin (parallel b-pleated sheet) [overhead, Figure 7-26].

3-D Structure of Proteins 3

Secondary Structure, cont.

Collagen strand: This is a specialized structure occurring in only a particular family of fibrous proteins. It does not occur in globular proteins that I am aware of.

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  Collagen triple helix. Note repeating sequence of -(gly-x-y)- where x is usually proline and y is usually hydroxyproline. (Fig 4.36) [overheads: 11-8&10, S; 4-10 to 12]
  

Non-repetitive secondary elements: Proteins can also have non-repetitive secondary structures which consist of a few residues in a turn or loop. Among these are:

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  beta-turns:
  
  • 
    Type I turns: Fig. 4.18, left [overhead 7.22, V&V] four amino acid residues in a 180° turn, usually H-bonded between the carbonyl O of the first residue and the amide N of the fourth. Proline is often the second residue. [overhead, 7-22 V&V]
    
  • 
    Type II turns: Fig. 4.18 [overhead 7.22, V&V] four amino acid residues in a 180° turn, usually H-bonded between the carbonyl O of the first residue and the amide N of the fourth. Glycine is most frequently the third residue and proline is often the second residue. [overhead, 7-22 V&V]
    
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  A partial turn of a 3₁₀ helix. Short sections of this helix often occur at the ends of alpha-helixes as transitional elements.
  

Tertiary Structures

The Tertiary structure describes the overall folding of a single covalent structure.

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  Lysozyme model [overhead, model]
  

As the number of known protein structures increased additional patterns became obvious within the tertiary level of structure: Motifs & Domains.

Super Secondary structures (Motifs)

Recall the two classical structures based on the beta-strand:

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  Anti-parallel b-pleated sheet: strong, linear H-bonds spaced adjacent, then R grp, then single, then R grp, then adjacent etc. (Fig 4.15b) [overhead 7-17 V&V, 5.19 P]
  
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  Parallel b-sheet: evenly spaced, but slanted H-bonds (less stable), (Fig 4.15a) [overhead 5.19 P]
  

Let's next look at some of the other more common motifs found in globular proteins (Fig 4.19 of your text):

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  Hairpin - b-strand-short loop-b-strand
  
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  b-meander - an anti-parallel beta sheet with short connecting loops
  
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  aa motif - two successive alpha-helixes with slightly inclined axis to give better contact between side chains
  
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  bab unit: alternate pattern of beta-strands and alpha-helixes
  
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  Greek Key
  
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  b-sandwich
  

Domains

Large proteins (>200 aa's) usually fold up in smaller pieces of 100-200 aa's called domains. Recall that we define a Domain as an independent folding region in a protein. Often defined by clefts in 3D structure giving globular elements connected by "hinges" (single strand segments connecting the domains). Domains have the advantages of speeding up the folding process (fold domains independently, then assemble resultant folded domains - effectively processing folding of domains in parallel). Another advantage of domain structure is that nature can take bits of DNA specifying particular domains with particular functions and assemble them in new combinations to get new activities (e.g. combine an ATP binding site and a sugar binding site to give a sugar phosphorylating protein).

Example: IgG , domains, exons and evolution. [overheads: IgG/proteins; 7.23 MvH]

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  IgG made up of four independently synthesized proteins, 2 heavy chains with 4 domains each, and 2 light chains with 2 domains each.
  
  • 
    Domain types: b-meander [anti-parallel b-sheet], b-barrel. (Note that Motifs and Domains often use the same nomenclature, and indeed often overlap. Can in fact have Motif = Domain = Tertiary structure!)
    
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  Domains correspond to exons of DNA (frequently, but not always the case)
  
  • 
    The domains are all apparently related through gene duplication in the remote past.
    
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  The active site of IgG (2/IgG) is made up between two domains, one from a heavy chain and one from a light chain.
  
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  When immune system is developing individual cells express single IgG molecules made from randomly expressed heavy and light chains.
  

In a similar manner we see that many enzymes have active sites created between two domains, often one domain binds one substrate while the second binds a second substrate.

Its as if these proteins were designed by taking "off-the-shelf" components, assembling them, and then over time (and generations) tuning the combination up.

3-D Structure of Proteins 4

Domains, cont.

Note that domains will have their own tertiary structures, made up of secondary and frequently supersecondary elements. Domains can be categorized into four main groups:

1. 
   All alpha
   
2. 
   All beta
   
3. 
   alpha/beta (have alternating alpha and beta structures, such as in the beta-alpha-beta motif)
   
4. 
   alpha + beta (local clusters of alpha and beta in same chain with each cluster consisting of contiguous primary structure).
   

Groups of motifs forming the core of the tertiary structures of domains are referred to as Folds. (p 99) Over 600 folds have been discovered, with an expectation that about 1,000 exist. (a bunch, but well below the infinite number possible!) Common examples include (Fig 4.24) [overhead]:

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  Parallel twisted sheet.
  
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  Beta barrel.
  
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  Alpha/Beta barrel.
  
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  Parallel twisted sheet .
  

Folds/Motifs are often more highly conserved than sequences, and so are used along with sequences to trace relatedness among molecules and thus organisms. An example of conservation for a domain is seen in Cytochrome c as shown in your text in Figure 4.21.

Quaternary Protein Structure

ternary (4°) structures (Fig. 4.25; overheads: MvH 6.26, Fig 25): Geometrically specific associations of protein subunits; the spatial arrangement of protein subunits.

Folding Hierarchy Overview

 

Rationale for quaternary: There are a variety of advantages to large structures:

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  Increasing the size of a protein allows better "fits" for catalysis and binding - many weak bonds are needed to maintain specific structures.
  
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  Can bring sequential active sites of metabolic pathways into close proximity.
  
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  However, large peptides have some problems:
  
  • 
    The process of folding slows tremendously with increasing size, thus folding individual subunits, and assembling these subunits can greatly enhance folding efficiency.
    
  • 
    Get about 1 error / 10³ aa residues due to the precision of the translation of messenger RNA to protein. Thus need to keep residue number down.
    
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  Interacting subunits provide mechanisms for regulation.
  

Quaternary structures allows the assembly of large to extremely large structures. 

 

Protein Folding

Primary structure specifies tertiary (& therefore quaternary) structure. This is known from in vitro denaturation/renaturation studies of small proteins.

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  Denaturation means to unfold to non-functional state, often achieve a "random coil" in solution,
  
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  Renaturation means to return to the properly folded, natural, and functional state.)
  

The classic study involved Ribonuclease: Reduce (break) -S-S- bonds, denature with urea to random coil. Now can renature by gently removing denaturant (urea) and oxidize -S-S- bonds. [overhead 5.41, P] Enzyme activity fully recovered. X-ray diffraction image same! Note - no gremlins, no magic, done in "test tube."

Other small proteins, such as Myoglobin and proinsulin, fold up spontaneously in the same manner as Ribonuclease. However, insulin fails to fold correctly, since a peptide essential to folding has been cleaved off.

Accesory Folding Proteins. The ribonuclease renaturation-type experiment has not been repeated with large proteins, which seem to require the participation of "folding catalysts," the chaperones, to aid their folding.

Modul 4. Enzymes.

The nomenclature and classification of ferments. Frame and catalytic properties of ferments. Temperature effect, рН, concentration of ferment and substrate for speed of enzymatic reactions. Regulation of activity of ferments
Enzymes are found all around us, they are found in every plant and animal. Any living organism needs enzymes for its functioning. All living being are controlled by chemical reactions. Chemical reactions that are involved in growth, blood coagulation, healing, combating disease, breathing, digestion, reproduction, and everything else are catalyzed by enzymes. Our body contains about 3,000 enzymes that are constantly regenerating, repairing and protecting us. 

Enzymes are powerhouses that are able to perform variety of functions in the human body. Enzymes are wondrous chemicals of nature. Enzymes are used in supplement form in medical arena. Although our bodies can make most of the enzymes, our body can wreak havoc the body's enzyme system and cause enzyme depletion due to poor diet, illness, injury and genetics.