Structural Hierarchy in Proteins - Biochemistry - Lecture Slides, Slides of Biochemistry

Download Structural Hierarchy in Proteins - Biochemistry - Lecture Slides and more Slides Biochemistry in PDF only on Docsity! Structural hierarchy in proteins Docsity.com Color conventions Docsity.com The Polypeptide Chain oO R H nm, | |° | io we cHy N HoOC ee ™ N ol ~~ Cc a Noy “a Ry ‘ 0 VJ Amina . terminus Peptide bonds Carhoxy terminus Docsity.com Chapter 5 Covalent structures of proteins Proteins function as: 1. Enzymes:biological catalysts 2. Regulators of catalysis-hormones 3. Transport and store i.e. O2, metal ions sugars, lipids, etc. 4. Contractile assemblies Muscle fibers Separation of chromosomes etc. 5. Sensory Rhodopsin nerve proteins Docsity.com 6. Cellular defense immuoglobulins Antibodies Killer T cell Receptors 7. Structural Collagen Silk, etc. Function is dictated by protein structure!! Docsity.com 3. Tertiary Structure 3 = the 3 dimensional structure of an entire peptide. Great in detail but vague to generalize. Can reveal the detailed chemical mechanisms of an enzyme. Docsity.com 4. Quaternary Structure 4 two or more peptide chains associated with a protein. Spatial arrangements of subunits. Chapter 5.3 is how to determine a protein’s primary structure. “Protein Chemistry” Docsity.com Example of each level of protein structure (UeqIx) esuncgns e6COuqsIA IUfO SU O|!RoWELIC bLoseiu OL EWOROpiU geecwpleq See fUG LON e6bsisse cugive © (B cysiu of yewoRjopiu) ONssewwsIA efincgnie: ous Cowbjese bioseiu CusiU IGLEISIA efincgnie: 8 () bUIWSIA efincsnie (SWJIUO ScIq 2e6dn6euUCE IU 9 bojAbebsiqe cugiu) Phe — IS — Hl2 — GIA — phe - phe - AS] - ren - GA yg - Docsity.com Bovine insulin: note the intra- and inter- chain disulfide linkages Docsity.com 2. Sequencing the peptide chains: a. Fragment subunits into smaller peptides  50 AA’s in length. b. Separate and purify the fragments c. Determine the sequence of each fragment. d. Repeat step 2 with different fragmentation system. Docsity.com 3. Organize the completed structure. a. Span cleavage points between sets of peptides determined by each peptide sequence. b. Elucidate disulfide bonds and modified amino acids. At best, the automated instruments can sequence about 50 amino acids in one run! Proteins must be cleaved into smaller pieces to obtain a complete sequence. Docsity.com Disadvantage with the Dansyl-chloride method is that you must use 6M HCl to cleave off the derivatized amino acid, this also cleaves all other amide bonds (residues) as well. Edman degradation with Phenyl isothiocyanate, PITC Docsity.com Edman degradation has been automated as a method to sequence proteins. The PTH-amino acid is soluble in solvents that the protein is not. This fact is used to separate the tagged amino acid from the remaining protein, allowing the cycle of labeling, degradation, and separation to continue. Even with the best chemistry, the reaction is about 98% efficient. After sufficient cycles more than one amino acid is identified, making the sequence determination error-prone at longer reads. Docsity.com Demonstration of Edman degradation Use your CD disk- install it and run chapter 5 Edman degradation. Docsity.com Amino acid composition The amino acid composition of a peptide chain is determined by its complete hydrolysis followed by the quantitative analysis of the liberated amino acids. Acid hydrolysis (6 N HCl) at 120 oC for 10 to 100 h destroys Trp and partially destroys Ser, Thr, and Tyr. Also Gln and Asn yield Glu and Asp Base hydrolysis 2 to 4 N NaOH at 100 oC for 4 - 8 h. Is problematic, destroys Cys Ser, Thr, Arg but does not harm Trp. Docsity.com Amino acid analyzer In order to quantitate the amino acid residues after hydrolysis, each must be derivatized at about 100% efficiency to a compound that is colored. Pre or post column derivatization can be done. CH CH O O + HS CH2 CH2 OH + H3N CH O R C O N CH O R C O CH2 CH2 OHS o-Phthalaldehyde (OPA) 2-mercaptoethanol Amino acid These can be separated using HPLC in an automated setup Docsity.com Amino acid compositions are indicative of protein structures Leu, Ala,Gly, Ser, Val, Glu, and Ile are the most common amino acids His, Met, Cys, and Trp are the least common. Ratios of polar to non-polar amino acids are indicative of globular or membrane proteins. Certain structural proteins are made of repeating peptide structures i.e. collagen. Docsity.com How to assemble a protein sequence 1. Write a blank line for each amino acid in the sequence starting with the N-terminus. 2. Follow logically each clue and fill in the blanks. 3. Identify overlapping fragments and place in sequence blanks accordingly. 4. Make sure logically all your amino acids fit into the logical design of the experiment. 5. Double check your work. Docsity.com 1 2 3 4 5 6 7 8 9 10 11 12 13 14 H3N-_-_-_-_-_-_-_-_-_-_-_-_-_-_-COO K F - A - M - K K - F - A - M Q - M - K D - I - K - Q - M G - M - D - I - K Y - R - G - M Y - R Cyanogen Bromide (CN Br) Cleaves after Met i.e M - X D - I - K - Q - M K K - F - A - M Y - R - G - M Trypsin cleaves after K or R (positively charged amino acids) Q - M - K G - M - D - I - K F - A - M - K Y - R Docsity.com There are a variety of ways to purify peptides All are based on the physical or chemical properties of the protein. Size Charge Solubility Chemical specificity Hydrophobicity/ Hydrophylicity Reverse Phase High Pressure Liquid Chromatography is used to separate peptide fragments. Docsity.com Malaria as Sickle-cell gene ® Docsity.com Deoxyhemoglobin aggregates and deforms cell. Primary structure changes dictate quaternary structure. Why did the problem not die out? Homozygotic Heterzyatic Homozygotic normal sickle cell trait sickle cell gets malaria resistant gets sickle cell to malaria dies dies Docsity.com Species variation in homologous proteins The primary structures of a given protein from related species closely resemble one another. If one assumes, according to evolutionary theory, that related species have evolved from a common ancestor, it follows that each of their proteins must have likewise evolved from the corresponding ancestor. A protein that is well adapted to its function, that is, one that is not subject to significant physiological improvement, nevertheless continues to evolve. Neutral drift: changes not effecting function Docsity.com Phylogenetic tree Indicates the ancestral relationships among the organisms that produced the protein. Each branch point indicates a common ancestor. Relative evolutionary distances between neighboring branch points are expressed as the number of amino acid differences per 100 residues of the protein. PAM units or Percentage of Accepted Mutations Docsity.com Birds Penguin Chicken, turkey Mammals i Fungi eee Pekin duck 3 Human, S D. klockeri Chimpanzee Monkey Pigeon Horse t " Snapping 14.5 Cow, pig, 3 Rabbit 6 Kangaroo/ .., turtle Reptiles Cas: sheep 5 me . kruset Dog 5 25 Amphibians oo Doe pulifod : Tuna i Plants 12.5 Insects 6 P Bonito mune been aor Carp Fish moth Drosophila 65 Bomwvorm , Screwworm °f 4 Dogfish 2 Baker's yeast moth 3 2 fly cen 2 Lamprey 75 = a Wheat Castor ‘ 13 4, eae Sunflower 14 73) 78 a 12 25 2 15 12 Docsity.com PAM values differ for different proteins. Although DNA mutates at an assumed constant rate. Some proteins cannot accept mutations because the mutations kill the function of the protein and thus are not viable. Docsity.com Hemoglobin: • is an oxygen transport protein •it must bind and release oxygen as the cells require oxygen Myoglobin: • is an oxygen storage protein •it binds oxygen tightly and releases it when oxygen concentrations are very low Docsity.com The globin family history 1. Primordial globin gene acted as an Oxygen-storage protein. 2. Duplication occurred 1.1 billion years ago. lower oxygen-binding affinity, monomeric protein. 3. Developed a tetrameric structure two a and two b chains increased oxygen transport capabilities. 4. Mammals have fetal hemoglobin with a variant b chain i.e. g (a2g2). 5. Human embryos contain another hemoglobin 2e2. 6. Primates also have a d chain with no known unique function. Docsity.com oa Hemoglobin family Man ae Man Prey Rhesus . Le Man =‘ monkey Man Man J B 6 B Cow ¥ f | ” J Primates ao Y Cow 1 5 / Myoglobins | Primates Platypus Man Cow ’ Y f / Platypus /,Mammals / ‘4 B Chicken , ean” Land Primates)// Chick \ “ Lan y e \ vertebrates / Platypus c Carp \ aul j jammals Carp a vertebrates Shark Chichen Shark fo ‘Land vertebrates Xs. Shark Lamprey 4 cae aes FP Lamprey BHemoglobin family _ Primordial globin Whale Docsity.com