Forces stabilising structure of proteins

covalent and non-covalent bonds

1. FORCES STABILISING STRUCTURE OF PROTEINS PRATHYUSHA Msc BIOTECHNOLOGY

2. Covalent bonds Peptide bond Disulphide bridges Non-covalent bonds Van der Waals forces Short range repulsion Electrostatic bonds Hydrogen bonds Hydrophobic interactions

3. COVALENT BONDS Covalent interactions (bonds) hold atoms together within molecules . Covalent bonds are stronger than molecular interactions. Covalent bonds break and/or form during chemical reactions. Bonds break during the oxidation of sugar to form carbon dioxide and water, which is a chemical reaction called fire. By contrast, covalent bonds remain intact and unchanged when (a) ice melts, (b) water boils, (c) proteins unfold, (d) RNA unfolds, (e) DNA strands separate, and (f) membranes disassemble. These processes are not chemical reactions. The processes (a-f) of melting, boiling, unfolding, strand separation and disassembly involve changes in molecular interactions.

4. COVALENT BONDS Peptide bonds between Amino Acids (C-N). Can be broken down into individual amino acids by hydrolysis with 6M acid/alkali, or by proteases/ proteolytic enzymes. Disulphide bridges form between cysteine to form cystine. (Cysteine has -SH which forms disulphide bridge -S-S- with another HS-). Bridges are broken down by reduction with β- mercapto ethanol to form cysteines once again.

5. MOLECULAR INTERACTONS Molecular Interactions are attractive or repulsive forces between molecules and between non-bonded atoms. Molecular interactions are important in diverse fields of protein folding, drug design, material science, sensors, nanotechnology, separations, and origin of life. Molecular interactions are also known as non-covalent interactions or intermolecular interactions. Molecular interactions are not bonds. All molecular interactions are fundamentally electrostatic in nature and can described by some variation of Coulombs Law.

6. Native states. In biological systems (i) proteins fold into globular structures called native states, (ii) ribosomal and transfer RNAs also fold into native globular structures, (iii) DNA forms double stranded helices, (iv) phospholipids form membranes, and (v) proteins assemble with DNA, RNA, membranes and with other proteins. These native states and assemblies are stabilized by molecular interactions of enormous number and complexity. Native states are destabilized by their low conformational entropy. Denatured states. When you unfold a protein or an RNA (denature them) or separate two strands of DNA (melt it), or disassemble and melt the ribosome, then interior regions become exposed to the surroundings, which are mostly water plus some ions. Molecular interactions within the native state or assembly are replaced by molecular interactions with aqueous surroundings.

7. A delicate balance. Molecular interactions stabilize both native and denatured states. Native biological macromolecules and assemblies are marginally stable. Biological systems are held in 'delicate balance between powerful countervailing forces'. A small perturbation can tip the balance between the folded and unfolded states. A small change in pH or temperature or a single mutation can unfold a protein.

8. Have you ever denatured a protein (converted it from the native state to denatured state)? Yes. When you heat an egg to around 60° C, the albumin proteins denature and aggregate. You are not breaking bonds when you boil an egg - you are changing and rearranging molecular interactions. The aggregated protein forms large assemblies that scatter light, giving the egg a white appearance.

9. NOTE ON VAN DER WAALS INTERACTIONS Molecular interactions were discovered by the Dutch scientist Johannes Diderik van der Waals . He noticed that molecules are sticky, like wet jelly beans. The phrase 'van der Waals interaction' has come to mean cohesive (attraction between like), adhesive (attraction between unlike) and/or repulsive forces between molecules. For our purposes, 'van der Waals interaction' is not sufficiently informative or descriptive or specific. The problem is that some people use 'van der Waals interactions' to describe the totality of molecular interactions but others use it to describe various subsets of molecular interactions. Here we avoid the term "van der Waals interaction" because it is not well-defined and does not decompose the interactions in a physically meaningful way.

10. SHORT RANGE REPULSION Atoms take space. Force two atoms together and they will push back. When two atoms are close together the occupied orbitals on the atom surfaces overlap, causing electrostatic repulsion between surface electrons. This repulsive force between atoms acts over a very short range, but is very large when distances are short. Short range repulsion is important to you. It prevents your hands from passing through each other when you clap, and prevents atoms from collapsing into tightly packed states.

11. In B-DNA, the distance between stacked base pairs is 3.4 Å as required by short range repulsion. Base pairs are slightly inclined relative to the helical axis; Base pair normals are not exactly parallel to the helical axis. Therefore the rise per base pair along the helical axis is slightly less than 3.4 Å.

12. ELECTROSTATIC INTERACTIONS Electrostatic interactions are between and among cations and anions, species with formal charge of ...-2,-1,+1,+2... Electrostatic interactions can be either attractive or repulsive, depending on the signs of the charges. Like charges repel. Unlike charges attract. The electrostatic interactions within a sodium chloride crystal are called ionic bonds. But when a single cation and a single anion are close together, within a protein, or within a folded RNA, those interactions are considered to be non-covalent electrostatic interactions. Non-covalent electrostatic interactions can be strong, and act at long range. Electrostatic interactions fall off gradually with distance (1/r, where r is the distance between the ions).

13. Electrostatic interactions are the primary stabilizing interaction between phosphate oxygens of RNA (formal charge -1) and magnesium ions (formal charge +2). There are many magnesium ions associated with RNA and DNA in vivo . Electrostatic interactions are highly attenuated (dampened) by water. In protein folding, RNA folding and DNA annealing, electrostatic interactions are dependent on salt concentration and pH. In RNA (for example in the ribosome), anionic phosphate oxygens (-1 charge) engage in attractive electrostatic interactions with cationic magnesium ions (+2 charge). Two phosphate groups can 'clamp' onto the Mg 2+ ion. The O to Mg 2+ distance is 2.1 Å. The dashed lines represent favorable electrostatic interactions.

14. ION PAIRS IN PROTEINS Favorable electrostatic interactions between paired anionic and cationic amino acid side chains are reasonably frequent in proteins. Ion Pairs, sometimes called Salt Bridges, are formed when the charged group of a cationic amino acid (like lysine or arginine) is around 3.0 to 5.0 Å from the charged group of an anionic amino acid (like aspartate or glutamate). The charged groups in an ion pair are generally linked by hydrogen bonds, in addition to electrostatic interactions.

15. An ion pair within a folded protein. An anionic aspartic acid (charge = -1) engages in attractive electrostatic interactions with cationic arginine (charge = +1). The dashed lines represent hydrogen bonds.

16. HYDROGEN BOND Attraction between hydrogen atom which is bonded to electronegative atoms such as F,O,N and an adjacent electronegative atom. Why hydrogen? Hydrogen is special because it is the only atom that (i) forms covalent sigma bonds with electronegative atoms like N, O and S, and (ii) uses the inner shell (1S) electron(s) in that covalent bond.

17. The most common hydrogen bonds in biological systems involve oxygen and nitrogen atoms as A and D. Keto groups (=O), amines (R 3 N), imines (R=N-R) and hydroxyl groups (-OH) are the most common hydrogen bond acceptors in DNA, RNA, proteins and complex carbohydrates. Hydroxyl groups and amines/imines are the most common hydrogen bond donors. Hydroxyls and amines/imines can both donate and accept hydrogen bonds.

18. An isolated ammonia molecule, just like a water molecule, can form strong hydrogen bonds with either hydrogen bond donors or acceptors. Ammonia is more basic than water, and therefore ammonia is a better hydrogen bond acceptor than water.

19. HYDROPHOBIC EFFECT The hydrophobic effect is the insolubility of oil and other non-polar substances in water. If you mix oil and water by vigorous shaking, you will observe spontaneous unmixing - meaning mixing entropy is negative. Spontaneous unmixing is strange and unusual. The unmixing of non-polar substances and water is the hydrophobic effect in action. Hydrophobic substances are those that are soluble in non-polar solvents (such as CCl 4 or cyclohexane or olive oil). Hydrocarbons (CH 3 CH 2 CH 2 .... CH 2 CH 3 ) are hydrophobic.

20. A very important factor to remember is that the hydrophobic effect is fully a property of water; it a consequence of the distinctive molecular structure of water and the unique cohesive properties of water. Hydrophobic substances are passive participants in the hydrophobic phenomenon. The driver for unmixing is interfacial water molecules (directly adjacent a hydrocarbon molecule or plastic surface) that maintain water-water interactions at the cost of rotational and translational freedom . Interfacial water has low entropy and is therefore unfavourable. Water gains entropy and therefore stability by minimizing the amount of interfacial water. This is why water droplets deform on a hydrophobic surface - droplets adjust their shape to minimize contact with a hydrophobic surface.

21. CONTER-ION RELEASE FROM NUCLEIC ACID Counter ions are released when a cationic protein binds to DNA. This release explains the dramatic salt dependencies of DNA-protein complexes. High salt destabilizes DNA-protein complexes. If the bulk salt concentration is low, the protein binds tightly to the DNA. If the bulk salt concentration is high, the protein binds weakly. Counter ion release explains much of the salt dependencies of DNA melting, RNA folding and DNA condensation.

22. Figure shows an axial view of DNA, represented as a anionic cylinder. Cationic counter ions (orange shading) surround the cylinder. The concentration of cations decreases with distance from the surface of the cylinder. The deeper orange shading indicates more concentrated cations. The panel on the right illustrates how both anionic counter ions (blue) associated with a cationic protein, and cationic counter ions (orange) associated with anionic DNA, are released to bulk solution when the protein binds to DNA. This release of counter ions drives the association.

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