Dana Alsulaibi. Jaleel G.Sweis. Mamoon Ahram

Transcription

1 15 Dana Alsulaibi Jaleel G.Sweis Mamoon Ahram

2 Revision of last lectures: Proteins have four levels of structures. Primary,secondary, tertiary and quaternary. Primary structure is the order of amino acids in a polypepetide peptide. It is very important as it determines the characteristics of the next levels. Secondary structure (alpha helix + beta strand) presents the localized organized structure (helix,sheet,strand,turn,loop ). Secondary structure is stabilized by the hydrogen bonds between the backbone groups ;where the carbonyl group is the H bond acceptor and the amino group is the H bond donor ( R groups have no influence on the secondary structure although some amino acids may disrupt the structure). Lecture 15: Parallel vs anti parallel beta sheets Anti-parallel beta sheets are more stable than parallel beta sheets. In anti parallel sheets, hydrogen bonds are formed between the amino acid in the first sheet and the amino acid directly opposite(above) to it in the second sheet. This provides stability unlike parallel sheets where each amino acid forms a hydrogen bond to the left or right amino acid of the opposite sheet(not with the one directly above it). (my understanding: carboxyl and amino groups are directly opposite to each other in the anti parallel order, making hydrogen bonds stronger) Plus, vertical angles are stronger than bent angles making them more stable.

3 *β sheets can form between many strands, typically 4 or 5 but as many as 10 or more *Such β sheets can be purely antiparallel, purely parallel, or mixed *Valine, threonine and Isoleucine tend to be present in β-sheets.however, Proline tends to disrupt β strands What is a turn(beta turn or hairpin bend)? Turns are compact, U-shaped, secondary and small structures which allow proteins to take a 3 dimensional structure by enabling them to bend and go back. These turns are composed of 4 amino acids. 2 of them are Glycine and Proline. Glycine is small and its r group doesn t create repulsion so It needs less space. As for Proline, its rigidity breaks the continuity of the peptide at the turn, allowing the peptide to make a turn. Turns are also stabilized by hydrogen bonds which are formed between two amino acids between their the backbone groups( carbonyl and amino groups). Super secondary structures They are multiple secondary structures that are formed together. motif domain

4 Two types 1) Motif( a module) : multiple secondary structures that exist consecutively in a poly peptide sequence. (no seperation between an alpha helix and the next alpha helix or beta sheet. They come directly after each other with other structures in between) Uses of a motif : helps to determine the protein structure only but not biological function. Examples of motifs : Helix- loop -helix Helix-Turn- helix *Turns are smaller than loops Binding proteins The helix-loop-helix and helix-turn-helix are simple motifs that usually serve as binding proteins in DNA.

5 Immunoglobulin module is an example of more complex motifs. Immunoglobulins are proteins that are produced by immune cells.they have very complex structure that is mainly composed of beta strands and sheets. Having a Y shape, the upper part binds to the antigen. Tertiary structure The 3d dimensional arrangement of all amino acids. The area they take and spatial arrangement they have (in space) after folding into a polypeptide. Different ways to look at proteins a) Trace structure: represents the backbone only with no apparent R groups b) Ball and stick model: the balls represent the location of atoms in space and their angles c) Ribbon structure: alpha helixes are represented as ribbons and beta strands are represented as arrows which helps determine the orientation of beta strands(parallel vs antiparallel) d) Cylinder structure: alpha helixes are represented as cylinders and beta strands are represented as arrows e) Space filling structure : occupied by atoms only with no spaces. Helps to )طعجات و زوايا) indentation look at the surface of the protein and any present

6 f) Protein surface map : to look at the topography (the outside structure) of the protein. Especially helps to determine the structure of drugs that can bind to the protein. In the will be these we them well. exam we asked about structures so should know Doctor Ma moon possible exam questions bring the structure and ask for its name Ask the number of alpha helixes and turns in a specific structure What is the name of the motif. Bonds that stabilize tertiary structure ( 4 non-covalent interactions between the R groups which determine protein structure): a) H-bonds: occur not only within and between polypeptide chains but with the surrounding aqueous medium.

7 b) Electrostatic bonds(charge-charge interaction) : occur between oppositely charged R-groups of amino acids. Also, it is called a salt bridge when it present in a protein structure. Ex, interaction between Na+ ion and Cl- ion is an electrostatic interaction Ex, lysine and glutamate by themselves(not residues) they can form electrostatic interaction. BUT when both are present as residues within a protein and they an electrostatic interaction then it is called a salt bridge * The same charged group can form either hydrogen bonding or electrostatic interactions. c) Hydrophobic : the most important one. Hydrophobic interactions fold toward the core of the protein to hide from water and expose the hydrophilic parts outside towards the aqueous environment. This starts determining the protein structure. It is also the most energetically favorable structure(requires minimum energy) However, some polar amino acids can exist in the core of functional proteins= enzymes. These enzymes need charged amino acids in the core for the reaction to take place inside of them. Can polar amino acids be found in the interior? Polar amino acids can be found in the interior of proteins In this case, they form H bonds to other amino acids or to the polypeptide backbone. They important roles in function of the protein d) Van der waals interactions: there are both attractive and repulsive van der waals forces that control protein folding. Although they are weak forces, they are significant because there are so many of them in large protein molecules. *the 4 previous interactions determine protein structure.

8 Stabilizing factors These factors do not affect the shape of the protein, but only stabilize it, preventing it from changing shape. 1 st factor : Disulfide bonds between two cysteine amino acids. If the bond was reduced and broken, the structure of the protein doesn t change, it only gets destabilized. The side chain of cysteine contains reactive sulfhydryl group(-sh) which can oxidize to form a disulfide bond(-s-s-) to a second cysteine.

9 The cross linking of two cysteines to form a new amino acid called cystine 2 nd factor : metal ions. These are non protein groups that can form either non covalent or covalent interactions with amino acids Non covalent examples: zinc with histidine, myoglobin/hemoglobin with iron(heme). Covalent bond examples : iron with histidine of the myoglobin/hemoglobin Function of previous non protein groups(stabilizing factors) : a)stabilize structure of proteins b) Provides the protein with its function Super secondary structures They are meltable secondary structures that are formed together. motif domain

10 Two types 2) Domain: Characterists of domains- Domains are larger than motifs, they are composed of hundred of amino acids residues in various combinations of(alpha helices, neta sheets, turns, and randon coils).they are also associated with function and structure of protein. If two proteins share a domain,these proteins have a similar function. Domains can fold independantly from the rest of the protein.if the domain was cut from its original protein,it will maintain its shape.genetic engineering consists of grouping 2 or more domains to make a final desired protein domains determine structure+ function of proteins domains may also be defined in functional terms: 1-enzymatic activity 2- binding ability (e.g a DNA-binding domain) Properties of proteins :denaturation and renatururation 1) Denaturation = unfolding of the protein by breaking of non covalent interactions(disruption of the native conformation of a protein) Complete denaturaion of a protein can only be achieved by applying a reducing agent to reduce and break the disulfide bonds. Otherwise, the protein can regain it s shape because parts of it are still connected to each other. Generally, the denaturated protein will lose its properties such as activity and become insoluble.

11 Denaturing agents Heat : heat breaks up Van Der Waals interactions by increasing kinetic energy and destabilizing atoms Extremes of ph: breaks H-bonds and electrostatic interactions. different Ph values effect protonation and deprotonation according to pka and thus effects the charges of amino acid side chains Detergents: disturb hydrophobic interactions. If a protein was put in a hydrophobic region it will flip to expose its hydrophobic regions and that may lead to its denaturation. Detergents ionic Non ionic e.g: Triton X-100(non-ionic, uncharged) sodium dodecyl sulfate(sds, anionic, charged) Denature the protein + add a charge

12 2)Renaturation Urea and guanidine hydrochloride disrupt hydrogen bonding and hydrophobic interactions. Reducing agents such as β-mercaptoethanol (βme) and dithiothreitol (DTT). Both reduce disulfide bonds. These do not denature the protein but destabilize it. Renaturation is the process in which the native conformation of a protein is re-acquired. The protein can fold back to its original structure by removing the denaturing factor and reducing agent. However mostly small proteins can do that. Large proteins need help. Renaturation can occur quickly and spontaneously and disulfide bonds are formed correctly. Factors that determine protein structure The least amount of energy needed to stabilize the protein. This is determined by: The amino acid sequence (the primary structure), mainly the internal residues. The proper angles between the amino acids The different sets of weak noncovalent bonds that form between the mainly the R groups. Non-protein molecules. Can an unfolded protein re-fold? If a protein is unfolded, it can refold to its correct structure placing the S-S bonds in the right orientation (adjacent to each other prior to formation), then the correct S-S bonds are reformed. This is particularly true for small proteins. The problem of misfolding

13 When proteins do not fold correctly, their internal hydrophobic regions become exposed.trying to hide from water they interact with other hydrophobic regions on other molecules, and form aggregates and clusters. Chaperons As we said before, large proteins need help in folding, this is done by chaperons( sheet. and will be discussed in the next )الرفيق/المساعد التوفيق كل

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