Polypeptides and Proteins (Week 3-4) Flashcards
(37 cards)
What is a protein?
-a long linear chain of amino acids
-myoglobin O2 binding protein from muscle with 153 amino acids in chain
How is myoglobin folded?
-compact 3-dimensional globular structure
-ribbon traces the path of the polypeptide backbone (no side chains just peptide bonds)
-blue at N-terminal progressing to red at C-terminal
-the ribbon is arranged in eight spiral or helical segments
What is a primary structure of a protein?
-the linear sequence of amino acids
-(myoglobin)
What is a secondary structure of a protein?
-regular repetitive patterns such as the helical sections in myoglobin that run along short sections of peptide chains
-might be asked to describe the organization of the backbone of the protein
What is the tertiary structure of a protein?
-the overall pattern of 3D folding of the whole polypeptide
What is the quaternary structure of a protein?
-still in linear form but will begin to fold on itself
-ex: the four globins in hemoglobin act cooperatively to improve oxygen transport
-interact with each other
How do you investigate the structure of a protein?
-find out which amino acids are present and in what sequence
-peptide bonds of protein are hydrolyzed with the help of catalyst to release individual amino acids
-must break peptide bonds for protein analysis (hydrolysis)
What techniques are used to break peptide bonds?
-acid hydrolysis: 6 M HCL 110 degrees 24-72 hour to completion
-base hydrolysis: 4 M NaOH 110 degrees 16 hour to completion
-Asn and Gln can be hydrolyzed and convert into Asp and Glu releasing them as ammonia
-after hydrolysis is complete can use chromatography to see how much amino acid is present
What are proteases?
-digestive enzymes
-proteins we eat get hydrolyzed in intestines to become amides
-catalyse hydrolysis of peptide bonds
How does chemical reactivity in hydrolysis and other biochemical reactions arise?
-from an unbalanced distribution of valence electrons
-C-C and C-H bonds share bonding electrons equally and are both non-polar and chemically unreactive
-hydrocarbons are non-polar and hydrophobic
-reactions involve atoms with unshared electrons (lone pairs ) or are electron deficient and pull electrons toward them due to electronegativity
(hydrolysis involves water and a catalyst breaking the peptide bond)
What is a nucleophile?
-what initiates biochemical reactions
-(nucleus lover) atom with a lone pair of electrons available to share
- seek out other groups that are electron-deficient (electrophiles which pull electrons towards or away from itself)
-water cant initiate hydrolysis alone because oxygen is weak
How does an atom with a lone pair use it?
- Hydrogen bond acceptor by attracting an O-H or N-H R-O
- Base if it captures H+
H+ + NH2-R—>NH3-R - Nucleophiles when it shares the lone pair with another electron-deficient atom to make a new bond
What is nucleophilic substitution?
-incoming nucleophile X attacks target atom C to displace leaving group Y which takes its bonding electrons away (electron-deficient atom is bonded)
-forming bond eliminates carbon
-X: + C-Y—–> X-C- + :Y
What is nucleophilic addition?
-in cases where Y is double bonded to C only one bond has to be given up so Y does not leave
X: + C=Y—–> X-C-Y
What is nucleophilic displacement?
-hydrolysis of peptide bonds
-C of the peptide bond is electron deficient (an electrophile) because the electronegative O bonded to C electrons pulls away from
-C takes up the incoming electron pair of OH in a new bond forming the transition state (semi-stable like compressed spring)
-amino N acts as leaving group allowing the peptide bond to break
How do you investigate the structure of a protein?
-identify the first amino acid by tagging it with fluorodinitrobenzene (bright yellow)
-fred sanger worked on insulin to break proteins to look at the amino acids as well as sequence proteins and DNA
-hydrocarbons are unreactive on some side chains
-the amino acid at the N-terminus of protein has a free alpha-amino group
-at high pH this group deprotonated to become NH2 a nucleophile
-reacts with fluorodinitrobenze HF is a good leaving group
NH3 protonated at pH 7 can not act as a nucleophile so you have to raise the pH to make it protonated
-hydrolysis releases the N-terminal amino acid with the yellow tag attached
-*hydrolysis destroys the rest of the polypeptide chain (sequence info is lost)
How do you determine the amino acid sequence of a protein?
-perh edman improved sanger’s method by allowing N-terminal amino acid to be reacted removed and identified without hydrolyzing the peptide bonds
-look at one amino acid at a time
-reaction can be repeated to identify amino acid #2 (no overlap)
What is Edman Degradation?
-involves coupling and cyclization 2 steps with never overlap
1. Coupling: requires base and the reaction must be complete before the next cyclization step can take place
2. Cyclization: requires acid that shares electrons with the first amino acid
2nd amino acid moves into 1st position
-reaction must be complete before the next coupling step can take place
-chain remains unbroken
-cycle can be repeated up to 50 times to give 50 AA sequence
-both steps happen under different conditions
-wont be asked to reproduce mechainism
What is selective hydrolysis?
-cuts the polypeptide at specific locations to yield a limited number of oligopeptides of definite size (protein chain is segmented)
-used to study long protein chains by cutting them into shorter oligopeptides
-the digestive enzyme trypsin binds and recognizes Arg or Lys side chains in peptides
-carboxylate group of the Arg or Lys is positioned next to catalytic unit of trypsin and is target for hydrolysis of peptide bond
How do trypsin and chymotrypsin convert polypeptides into smaller fragments?
-trypsin acts on Gly—Lys-X—Arg-Y—Lys-Pro—-Asn to yield Gly—Lys + X—Arg + Y—Lys-Pro—-Asn>at original C terminus end
-lys and arg are cut and hydrolize at C-terminus
-pro residue in the positon after Lys or Arg has wrong shape to fit the emzyme so no hydrolysis occurs if target Arg/Lys
-all fragments will have at the C-terminal end except the fragment from C-terminus
-chymotrypsin acts on Gly—Phe-X—Trp-Y–Phe-Pro—Tyr-Z—-Asn to yield Gly—Phe + X—Trp + Y–Phe-Pro—Tyr + Z—-Asn
-all fragments except C-terminus have Phe/Tyr/Trp at the C-end
What is cyanogen bromide?
-a chemical reagent which cuts polypeptide chains at methionine residues (another way to hydrolyze proteins)
-Br-C=N attacks S atom of Met which cannot bind with cysteine
-peptide chain is broken on carboxylate side and Met is converted to homoserine Hse (product of reaction) (serine with extra CH2)
-acts on Gly—-Met-X—Met-Pro—Asn to yield Gly—-Hse + X—Hse + Pro—Asn
-all fragments except C-terminus have Hse at C-end
How can mass spectrometry and chromatography separate proteins?
-this is a definitive means of identifying a known protein
-in experiments to determine the amino acid sequence of an unknown protein oligopeptide fragments from selective hydrolysis are first separated by chromatography and then sequences by edman’s method
What is the overlap method?
-peptide sequences are reassembled into a complete sequence
-two samples of the original polypeptide are each cut separately using two hydrolysis methods each targeting different sites
-sequences from one set of oligopeptides are lined up to overlap with oligo peptides from the other set to deduce how they were originally joined
What peptides are obtained using trypsin?
-A MET ASN LYS
-B ASP ILE ALA LYS
-C GLU ALA LEU PHE ARG
-D GLU LEU TYR GLN GLY
-C-terminal fragment always ends with TYR
