Proteins Flashcards
What does the primary structure consist of?
-the amino acid sequences -> help needed to fold correctly: thermodynamically or by chaperons
How are alpha helixes and ß-sheets obtained,
how are they held together?
-amino acid sequences linked together through H-bonds
How are tertiary structures formed?
-alpha helices and ß-sheets form long-distance interactions (Van der Waals, hydrophobic interactions, S-S bridges)
How are quarternary structures formed?
several protein subunits (1 subunit -> 1 amino acid chain) forming a multimeric structure (through H-bonding and Van der Waals)
Which amino acid doesn’t have a chiral C-atom?
Which isomer of amino acids is predominantly present in the body?
-Glycin
-L-isomer
Why is H-bonding between a carbonyl and a distal amino group in a chain possible to form alpha helices or ß-sheets?
-electrons in a chain are shared between the carbonyl and the nitrogen -> delta negative charge on the oxygen and delta positive charge on the H of the amino group -> making it possible for the O to share an electron with a distal H, and for the H to pick up electrons from a distal O
How is the alpha-helix structure held together between amino acids?
-1st carbonyl O (C=O) in the first amino acid forms an H-bond with the peptide H (NH2) of the 5th amino acid in the sequence (short-distance-interaction) -> 2nd carbonyl O with 6th h
-H-bonds stabilize the alpha-helix
-typically 11 AA long, 3.6AAs /turn
Why are alpha-helices formed by amino acid chains spontaneously?
-Because the alpha-helical for is stabilized by the H-bonds (1st to 5th; 2nd to 6th) and energetically favored
What are examples of alpha-helical structures?
-alpha keratin in sheep wool and collagen, human hair
-actin/myosin
How are ß-sheets formed?
-H-bonding between parts of the peptide that might be far from each other (long-distance interactions)
-Example: Beta-keratin in fingernails
-ß-sheets in the same direction are called parallel -> opposite direction anti-parallel
Why is helical wheel analysis useful?
Helps to predict polar and nonpolar side chains and therefore the function of the domain
-> hydrophobic: transmembrane domain (structural)
-> hydrophilic: extracellular or intracellular (signal-transduction)
What are the different interactions that help to shape a tertiary structure?
-Hydrophobic interactions (Tyrosine, Tryptophane, Phenylalanine
-Hydrophilic interactions: hydrogen binding, electrostatic (positive -negative (ionic) interaction)
-Thiol groups of cysteine form disulfid-bridges (when oxidized)
-interaction between acids and bases (ionic; ion-ion)
How are disulfide bridges different from other interactions, and what do they indicate?
-disulfide-bridges are covalent bonds (strong bonds) between cysteines
-provide rigidity to the structure
-inside the cell, there is not much free oxygen to oxidize the SH group to a disulfide bridge -> so the SH or S-S state indicates if the domain is intercellular or extracellular
Why are proteins sensitive to environmental factors?
Because the structure is held together by weak interactions that can be broken up when temperature, pH changes
How is it possible to have such a variety of gene products?
alternate splicing
-post-translational modifications -> carried out in the Golgi complex
What does protein stability refer to?
-determines if a protein will be in its native (active) or denatured (inactive) form
-affects the amino acid sequence as well as the 3-dimensional structure
-refer to thermodynamically and chemical stability
What are Biochemical reactions affecting protein stability?
Deamidation of Asn/Gln (Asn more vulnerable)
-most common
-self-attack -> the carbonyl (nucleophilic) on the R group is getting attacked by the backbone of the protein (NH)
-> 2 possible products -> cyclic intermediate Succinimide -> IsoAsp or Asp
-exchange from a polar group (Asn) to an ionizable functional group (IsoAsp or Asp) -> 3(Asp) : 1(IsoAsp)
Biochemical reaction affecting protein stability (2):
Pyroglutamate formation
Pyroglutamate formation (pyro = forming a cyclic structure)
-glutamic acid -> pyroglutamate (dehydration reaction)
-can be enzymatically or spontaneous
-occurs at N-terminal glutamate
-changes the charge of the protein
Biochemical reaction affecting protein stability (3):
Glycation
Glycation: similar -> similar to adding sugar to hemoglobin (HbAc1)
-addition of sugars (glucose, fructose) to Lys residues
-happen during manufacturing or in the bloodstream
How does Glycation on Lys affect the protein?
-loss of the positive charge bc the sugar is added to an amino group
-an increase in the size
How can the protein be monitored for changes and stability?
-the technique has to be able to detect the size, 3-dimensional structure, change in charge, polarity
What are the factors that affect chemical and physical stability?
pH, temperature, surface interaction, impurities/contamination
What are techniques to isolate proteins from the formulation or growth media, the cell
-Chromatography (LC or HPLC): separates compounds based on size and polarity -> data shown in the chromatogram (peaks for each protein -> representing different proteins with different sizes -> for proteins with the same size it would be a problem)
-Electrophoresis: separating based on the charge and somewhat the size by applying a current to the sample -> migration through the magnetic field -> positive proteins will migrate more to the negative side
Techniques for protein detection:
Infrared spectroscopy
After the separation of the proteins they need to be identified
-Fourier Transform Infrared Spectroscopy (FTIR) -> provides information about 1° and 2° structure (each peak corresponds to a behavior of a functional group) -> that pattern (peaks) is the fingerprint of the protein which can be matched to a database and compared to identify the protein
Techniques for protein detection:
Mass spectroscopy (MS)
-provides information about the mass and charge of the compound -> indicated in a ratio m/z
-to check if the cells are producing the preferred protein -> the cell has to be lysed and the protein mixture needs to be separated through chromatography (or gel electrophoresis) -> the single peptides are getting ionized (electrospray) so that they can be measured in the Mass spectroscopy
Process of Mass spectroscopy:
ionized peptide enters the mass analyzer -> provides peaks (m/z on the x-axis) -> in the next chamber they get hit by a gas (argon or neon) and are fragmented into even smaller pieces -> and undergo another round in the mass spectroscopy (MS2) -> with the difference between the peaks the weight of amino acids can be calculated and the AA can be identified
Why is it so hard to reproduce the same exact protein?
-20 different amino acids
-each of those can undergo post-translational modification
-200 types of modifications identified
Most common Post-translational modifications
-Phosphorylation: by Kinases on OH group of Serine, threonine, tyrosine -> change from polar to a charged residue -> changes the shape in 3D
-Glycosylation: forming glycans -> adding sugar residues to terminal NH2 (N-glycosidic - N-glycan) or OH (O-glycosidic - O-glycan) -> affects the charge and the size
What does a mass spectrometer need to detect?
-Large molecular size
-post-translational modifications: m/z -> the mass of the modification can be detected and identified (database) + charge can be detected to identify the modification
-measure biological material: bc proteins are in cells in growth media and are separated from those
