Introduction to Proteins and Protein Structure: E2

Functional Design of Proteins:

Proteins are made up of amino acids and its sequence gives proteins their diverse range of function.

Protein function involves physical changes in the 3D structure (conformation), due to various different bonding

The main function of proteins is to bind a range of molecules

-binding is characterised by two properties: affinity and specificity - important in enzymes and sometimes in immune systems

Affinity - strength of the bonding

Specificity - does it fit properly, does it have the specific active site

-enzymes are proteins that are highly efficient and specific catalysts

-protein receptors in the cell membrane, sense and transmit signals for transport

-antibodies are part of our defence mechanism which act to recognize and help destroy foreign bodies

Some proteins contribute to cell structure, organization, biomechanics and carriers of molecules e.g oxygen

Proteins can be modified by addition of other biological molecules:

  • protein + carbohydrate = glycoprotein - important in cell function and signalling

  • protein + lipid = lipoprotein

  • other modifications include phosphorylation/ de-phosphorylation etc

Amino Acids:

There are 20 standard amino acids which are common to all species

Structure:

Amino acids have a central carbon atom (alpha carbon) with 4 groups attached:

  • Primary amino group (-NH2)

  • Carboxyl group (-COOH)

  • Hydrogen atom

  • Variable ‘R’ group or side chain

All amino acids, except glycine, are enantiomers

Four groups arranged tetrahedrally around the alpha-carbon

Chirality - 2 non-superimposable mirror images; enantiomers or stereoisomers

-characterised by optical rotation of plane polarized light

-dextrorotatory (D; right) or laevorotatory (L; left)

L-amino acids are predominantly found in proteins

D-amino acids are found in bacterial cell wall and some antibiotics

Glycine ‘R’ group is hydrogen therefore, does not exist as a pair of stereoisomers and is a symmetrical molecule

R side chains vary in size, shape, charge, H-bonding capacity, hydrophobicity and chemical reactivity

R Side Chains:

The R side chains determine whether amino acids are hydrophilic or hydrophobic

Hydrophilic Amino Acids:

-are classified according to their charge at a neutral pH

  • basic - positively charge, amino groups

  • acidic - negatively charged, carboxyl groups

  • polar - uncharged at neutral, negative and positive charges are equal

In solution, amino acids exist in the neutral zwitterion form and depending on the pH, they can become an anion or cation

-as the pH decreases, a H+ ion will be added to the carboxylate

-as the pH increases the H+ will be removed from the NH3+

The 20 Amino Acids and their Properties

Non-polar, Aliphatic Amino Acids

These amino acids have hydrophobic interactions in protein structures

As glycine is small, it allows high flexibility however, proline confers enhances rigidity to protein structure

Aromatic Side Chains:

These are hydrophobic, but the hydroxyl group of tyrosine can form H-bonds - important in enzyme activity/signalling

Polar Uncharged Amino Acids:

Tend to be hydrophilic and are often important in enzyme activity - also influences protein structure

Cysteine undergoes di-sulphide bond formation due to being readily oxidized

Peptide Bonds:

Peptide bonds connect amino acids into linear side chains - primary sequence

Peptide bonds are covalent - amino group of one amino acid joins to the carboxyl group of another

-condensation reaction - one water molecule is removed

-as more amino acids are joined, they form a polypeptide - R groups usually on opposite sides of the peptide bond

Trans Configuration:

R side chains on alternating sides

Cis Configuration:

R side chains on the same side

  • The peptide bond is planar, restricting movement of the backbone of proteins

  • Torsion angle is the angle between groups on either side of a rotatable chemical bond:

  • alphaC - N bond is called Phi

  • -alphaC - C bond is called Psi - allows rotation

These rotations determine/drive protein folding and how variable side chains interact with each other

Levels of Protein Structure:

Primary:

The linear sequence of amino acids - polypeptide

Secondary:

Interactions within the polypeptide chain - a-helix or B-pleated sheets

Alpha Helix:

  • H-bonding between carboxyl and amino groups with distinct spacing

  • Stabilized by H-bonding between carbonyl of first and amino group of the fifth amino acid in helix and then second with sixth etc

  • Cylindrical, rod-like structures with R groups all positioned on the outside of the helix

  • Right handed helix - clockwise

  • Proline: distinct H-bonding pattern and cannot contribute to alpha helix structure

Beta ‘Pleated’ Sheet:

  • Repetitive H- bonding between adjacent sections of the polypeptide

  • Polypeptide sections run in the same direction - Parallel B sheet

  • Polypeptide sections run in opposite directions - Anti-parallel B sheet

  • R side chains protrude above and below the plane of the sheet

Connecting Loops - Coils:

  • Not repetitive, containing fewer backbone H-bonds

  • Sections that connect the regular structure of helices and sheets

Super-secondary structure - two or more alpha helices or B pleated sheets interacting with each other

Motifs/Folds - simple arrangement of structures that occur in more than one protein

Tertiary:

Overall, 3D arrangement of the polypeptide chain

-also includes details of binding of any prosthetic groups e.g. Haem

Determined by the amino acid sequence and properties of the R groups

Domains: distinct regions with specific structure which aids a specific function e.g enzyme binding a substrate

Stabilised by non-covalent, H-bonds, hydrophobic interactions, ionic interactions, electrostatic interactions, covalent, Van der Waals etc

Quaternary:

Interaction of two or more polypeptide chains into a multi-subunit complex

-multitude of bonding helps maintain protein 3D structure and stability including: non-covalent, covalent, di-sulphide, Van der Waals etc

Homomeric - identical polypeptide chains

Heteromeric - different polypeptide chains

Primary Sequence Dictates Sequential Folding:

Folding is determined by their amino acid composition

Secondary structures often form spontaneously, but the full 3D tertiary structure does not - accessory proteins are requires to assist the process of folding - complex and always spontaneous

Proteins are unfolded and refolded during movement through some organelles like the Golgi Body

Errors in protein folding can contribute to disease e.g amyloid plaques in Alzheimer’s

Formation of Disulphide Bonds:

Covalent bond which forms between cysteine residues - closely located with each other in the final conformation, but can be separated by amino acids

Facilitates intra and inter-molecule bonding

Function to stabilize the overall 3D structure

Formed under oxidizing conditions in the ER and are mainly found in secreted proteins and proteins of the extra-cellular matrix

Structural Motifs/Folds:

Super secondary structures

They are the interactions between 2 or more alpha helices and beta pleated sheets

Domains:

Represent larger recognisable regions of proteins

  • Functional Domains - mediate the function of the protein e.g. ability to bind to DNA

  • Structural Domain - 40 or more amino acids that form a stable secondary and tertiary structure - usually domains can fold into this structure independently of the rest of the protein

  • Many larger proteins have several recognisable structural motifs and domains, which also occur in other proteins with small variations and different combinations - modular nature of proteins

  • Proteins with similar sequences , closely related domains or similar domain structure are called protein family, more distant - superfamilies

Structural Classes of Proteins:

Globular proteins - high water solubility, compactly folded - enzymes and transporters like haemoglobin, growth factors, cytokines etc

Fibrous protein - elongated proteins, low water solubility, large amounts of regular secondary structures - often form stiff multimeric fibres e.g collagen, elastin, keratin

Collagen:

  • triple, left-handed helix

  • each polypeptide has a regular repeated amino acid sequence of ‘Gly-Pro-’X’

  • many triple helix molecules pack together to form fibres

Integral membrane proteins - associated with membranes, usually have alpha helices containing hydrophobic amino acids that span the hydrophobic , lipid region of membrane - includes receptors, transporters, cell-cell, cell-matrix problems

Importance of Protein Structure for Drug Development:

Aspirin covalently binds and inactivates cyclooxygenases which produce prostagladins and contribute to the sensation of pain associated with tissue inflammation/damage

Degradation of Proteins:

Proteins with well structured domains are difficult to access for proteases

Most proteins therefore are degraded by ubiquitin-proteasome pathway

Multiple copies of the small protein ubiquitin are coupled to a lysine residue in the protein to be degraded by specific ligases - process called polyubiquitination

Large protein complex called proteasome recognises the polyubiquitin chain, unwinds the secondary structure of the ubiquitinated protein and hydrolyses it into small peptides

Biomarkers:

Are used to detect tissue damage/disease

  • myocardial infarction damages the myocardium

  • result in release of cardiac muscle proteins into circulation, can be detected and gauge severity of damage

  • creatine kinase - involved in ATP synthesis

  • troponin - sarcomere protein, interacts with actin