Amino acids, peptides, proteins Flashcards
what properties make amino acids well suited to carry out a variety of biological functions?
capacity to polymerize
useful acid-base properties
varied physical properties
varied chemical functionality
what features do amino acids all share?
amino group, carboxyl group, alpha H, and a side chain R group which varies
all amino acids are chiral except ? What conformation are amino acids?
glycine. proteins only contain L amino acids
remember how to determine R vs L?
slide 8.
draw Fischer projection. if amino is right R, if amino is left L. Based on glyceraldehyde
non polar R group amino acids
glycine, alanine, valine, leucine, isoleucine, proline, methionine. be able to draw
polar R group amino acids
serine, threonine, cysteine, asparagine, glutamine
negatively charged R group amino acids
glutamate, aspartate
aromatic R group amino acids
phenylalanine, tyrosine, tryptophan
positively charged R group amino acids
lysine, histidine, arginine
what are uncommon amino acids? how are they created?
arise by post-translational modifications of proteins. reversible modifications, especially phosphorylation, are important in regulation and signaling
what is ionization of amino acids in acidic, neutral, and basic pH?
acidic: carboxyl and amino group protonated. amino acid is cationic
neutral: carboxyl group deprotonated and amino group protonated. net charge is zero (zwitterions)
basic: amino group is neutral (deprotonated) and carboxyl is deprotonated. amino acid is anionic
how do the carboxy and amino groups in amino acids compare to carboxylic acids and amines?
both are more acidic. Carboxyl groups in amino acids are much more acidic and amino groups in amino acids are slightly less basic.
how do amino acids act as buffers differently?
they have two pKa values, one for the carboxyl group and one for the amino group. this means they can buffer in two pH regions. remember that buffering is best when pKa = pH
how to calculate isoelectric point?
pI = pK1 + pK2 / 2
basically the average of the pKa’s
if the side chain is charged:
identify species with net zero charge (draw each ionization state) and identify the two pKa’s that determine the acid and base strength at this state. average those.
how are peptide bonds formed? draw a peptide bond
the carboxyl OH of one acid and the amino H of another act as leaving groups (water) and the peptide bond formed between the carboxyl C and the amino N
what are some functions of peptides?
hormones and pheromones
antibiotics
protection (toxins)
methods of protein separation
column chromatography: protein sample (mobile phase) moves through solid matrix (stationary phase) and separates as it moves down. general idea behind all of the other types
Ion exchange chromatography: separates by charge.
size exclusion chromatography: separates by size
affinity chromatography: separates by affinity
how does ion exchange chromatography work?
proteins in solution have different charges at the pH used. the stationary phase matrix consists of charged beads called exchangers (cation exchangers have - charge and bind cations, anion exchangers have + charge and bind anions). proteins with same charge as the exchangers elute faster while opposite charges interact with exchangers and are slowed.
how does size-exclusion chromatography work?
proteins in solution have different sizes. the stationary phase matrix consists of beads with small pores that trap smaller proteins as they elute. larger proteins are not slowed by traveling through the bead pores and elute faster
how does affinity chromatography work?
the stationary phase matrix is created to have a strong affinity ligand for the protein of interest and binds the protein while other proteins elute out. Once other proteins have eluted, the desired protein is freed from the matrix by introducing a ligand that binds to the protein, replacing the bond the protein had with the matrix. The ligand bound protein elutes
which amino acids can be observed via UV spectrophotometry?
aromatic amino acids. especially tryptophan and tyrosine, phenylalanine to a lesser extent
what is SDS and what’s it used for?
sodium dodecyl sulfate is a detergent that binds to and unfolds all the proteins, giving them a uniformly negative charge. this is important for gel electrophoresis because it removes the effect charge and native shape would have on movement, leaving only size as a factor
how can you determine pI of a protein experimentally?
Isoelectric focusing. a protein sample is applied to one end of a gel strip with a pH gradient and charge field. you want the - charge at the high pH and + at the low pH. the protein will be in a deprotonated (-) state at high pH and will migrate towards the + charge. as it migrates, the gel pH changes and once the pH causes a neutral ionization state (the isoelectric point) the protein will stop migrating and we can see the pI
do you remember how isoelectric focusing and SDS-PAGE gel electrophoresis can be used together?
slide 45
what is the definition of enzyme units, activity, and specific activity?
1 unit of enzyme = amount of enzyme required to convert one umol of substrate/min in optimal conditios
activity = total units of enzyme in solution
specific activity = # of enzyme units/mg total protein
how can we sequence proteins?
actual sequence is generally determined from DNA sequencing. 2 other methods:
Edman degradation: successive rounds of N-terminal modification, cleavage, and identification. only good for short chains around 20 rounds
Mass spectrometry: can precisely identify the mass of a peptide and thus the sequence.
remember how to display consensus sequences? How to read the notations [] {} () and so on?
slide 48
