Lecture 4 & 5 Flashcards
Who was Gerhardus Johannes Mulder?
-calculated molecular formula of egg and serum albumin
-proposed that proteins were made of smaller building blocks (called grundstoff)
-used acid hydrolysis to try to determine what grundstoff (discovered aa)
-first to use the word “protein”
Who was one of the fathers of biochemistry and what did he do?
Anselme Payen
discovered the first enzyme (diastase) in 1833 -extracted from malt solution
DID NOT KNOW AMYLASE WAS A PROTEIN
isolated and named cellulose
In what order are amino acid sequences written?
written from the N terminus (+NH3) to the C terminus (COO-)
What is the primary structure of proteins?
the amino acid sequence
What did Sanger’s work do?
implied there was a template in the cell that contained this information (the terminus and how they should be read)
What is the amino acid sequence?
Asp-Phe-Ile-Asn
or
DFIN
Why did Frederick Sanger win the Nobel Prize in 1958? What else did he do?
For sequencing insulin
-proved proteins had a defined primary structure
-second Nobel prize for sequencing DNA
What did Sanger use for his work?
Edman’s protein sequencing
What does cyanogen bromide (CNBr) cleave and what are the results?
cleaves the polypeptide at the C-terminal side of methionine
-produces a peptide homoserine lactone
-generates a new N-terminus
What is cleaved and what is blocked in protein sequencing?
cleaving disulfides
blocking cysteines
What is the order of the central dogma?
DNA makes RNA which makes Protein
Most polypeptides contain how many amino acids?
between 50 and 2000 amino acids
What is the average molecular weight of an amino acid?
110 daltons
What is the average molecular weight of proteins?
from 5500 to 220,000 daltons
Intracellular proteins
often lack disulfides
Extracellular proteins
often have disulfides
How can you get an estimate of a protein’s molecular weight?
by multiplying the number of protein amino acids by 110
Who received the Nobel prize for hemoglobin?
Max Perutz
Proteins are made by:
amino acid condensation
If we mix 2 amino acids in solution will we get a dipeptide?
no
Phylogenetic trees based on protein homology are consistent with _______ ____ sequences or morphology.
Phylogenetic trees based on protein homology are consistent with RIBOSOMAL RNA sequences or morphology.
What kind of proteins have a common ancestral protein?
homologous proteins
Conserved residues generally have a purpose related to:
structure
function
Proteins with similar ____ normally have similar _____.
Proteins with similar FUNCTION normally have similar STRUCTURE.
What is more conserved than sequence?
structure
Can proteins be modified?
yes
Insulin maturation involves what?
involves the formation of three disulfide bonds and three proteolytic cleavages of the peptide bond, resulting in two disulfide-linked polypeptides
For protein folding, what must there be with amino acids?
a. the stereochemistry of each amino acid must be maintained
-atomic connectivity
-bond lengths
-bond angles
-ring planarity
-dihedral angles
b. covalent bonds can NOT be broken or formed
What does the peptide plane restrict?
restricts torsions
In protein folding, what is Levinthal’s Paradox?
given a polypeptide of 101 residues with each residue having 3 possible conformations
-it would take YEARS to try all possible conformations…much longer than the universe has existed
What is a paradox?
folding for most small proteins is very fast
Anfinsen’s Dogma
for small globular proteins:
-native structure determined ONLY by the protein’s sequence
or
-native structure is a UNIQUE, STABLE, and KINETICALLY ACCESSIBLE minimum of the free energy for a given environment (temp., solvent concentration etc.)
Why was Christian B. Anfinsen awarded the Nobel prize?
Ribonuclease A folding
In the statement- “native structure is a UNIQUE, STABLE, and KINETICALLY ACCESSIBLE minimum of the free energy for a given environment (temp., solvent concentration etc.)” explain unique, stable, and kinetically assessable.
UNIQUE: only one conformation having this free energy
STABLE: conformation resists change (energy surface that looks like a funnel)
KINETICALLY ASSESSABLE: smooth energy path between the two states
Primary structure determines tertiary structure.
Afinsen’s Dogma
Disulfide bonds form AFTER the protein folds.
Protein Disulfide Isomerase (PDI)
How many possible S-S combinations are there?
105
What is denaturation?
the disruption of native conformation of a protein, with loss of biological activity (use heat or chemicals)
-proteins fold cooperatively
During the folding process, what happens to the polypeptide?
the polypeptide collapses into an intermediate “molten globule” due to the hydrophobic effect, then backbone is rearranged to achieve a stable native conformation
What is the driving force of protein folding?
-the large increase in entropy as water is released to bulk solvent
-hydrophobic collapse and secondary structure forms at same time
What are the four hypothetical protein-folding pathways, and which is which according to the picture?
1) extended structure
2) partial secondary structure
3) approximate tertiary structure
4) native structure
What do proteins fold by rather than by random?
Proteins fold by the progressive stabilization of intermediates rather than by random search
Chaperonin-assisted protein folding
needs energy
-hydrolysis of several ATP molecules is required
E. coli Chaperonin (HSP60 or GroE)
prevents incorrect protein aggregation
-protein folding takes place inside the central cavity
What are the four principles of the protein folding problem?
1) bury hydrophobic groups
2) expose charged groups or neutralize with salt-bridges
3) expose polar groups or satisfy h-bonding
3) obey stereochemical restraints
Hydrophobic collapse
results in the disordered peptide condensing around a weakly ordered folding nucleus, usually a small turn or short stretch of local secondary structure
Some folding features are cooperative, what is an example of this?
the amphipathic helix
What does an amphipathic helix need?
needs other structure to shield its hydrophobic surface
needs another HYDROPHOBIC structure to shield its HYDROPHOBIC surface
What is the problem of main chain h-bonding?
how to bury hydrophobic side-chains in the core of a protein and still satisfy the strong dipole and h-bonding needs of the peptide backbone
What did Linus Pauling the “Super Scientist” earn his two Nobel prize’s for?
1st: Helices and Strands
2nd: Peace Prize
The alpha-Helix
carbonyl (C=O) of reside 1 accepts a proton from the amide hydrogen (N-H) of residue (n +4)
3.6 residues per turn
H-bond between atoms 1 and 13
What does 3.6 residues imply?
For every turn (3.6 residues) of the alpha-Helix, there are between 3 and 4 H-bonds
Where are amphipathic helix’s typically found?
often found on the protein’s surface or at protein-protein interfaces
Is there such thing as a single amphipathic helix in solution?
NO
What do you do at the generate wheel? Helical Wheel
its where residues are spaced 100 degrees apart
-place AA at each position
-shows that the helix has one hydrophilic side and one hydrophobic side
What two ways can sheets and strands be? What is a, and what is b?
Parallel and anti parallel
a) anti parallel
b) parallel
With peptide bond torsion angles, how is native conformation achieved?
by rotating the main and side chain torsion angles
There are cis and trans isomers. There is a strong preference for ___ due to steric clash.
trans
Proline is a special case when it comes to cis and trans isomers. Why?
it permits both cis AND trans conformers
What two amino acids often appear in turns, and why?
GLY and PRO
GLY- small side chain
PRO - cis configuration is amenable to tight turns
For both type 1 and type 2 beta turns:
- the 1st and 4th amino acids are H-bonded
- the 2nd and 3rd are usually H-bonded to H20
Why is proline known as a helix killer?
it does not have an N-H group needed for continuing the H-bonding scheme
its unique cyclic structure prevents it from forming the necessary H-bonds to maintain a stable alpha helix causing kink or disruption breaking the helix structure
The pI of the protein depends on what?
its sequence
What is the pKa of the:
N-terminal
C-terminal
Ionizable side chains
N-terminal (NH3): ~ 10
C-terminal (COO-): ~ 2
Ionizable side chains: varies by a.a.
Proteins are charged. The charge depends on what?
the pH
as you change the pH of the buffer, the charge of the protein will also change as charged groups are titrated
The pH where the protein has no net charge is what?
is the pI of the protein
What are prions? Mis-folding
same sequence different fold
What do prions lead to?
leads to aggregation (plaque formation)
Building the proteins tertiary structure:
Motifs -> Folds -> Domains -> Structure
In: Motifs -> Folds -> Domains -> Structure
What are Motifs?
recognizable combination of secondary structures (helices, stands, and turns) that appear in different proteins and are usually associated with a function
In: Motifs -> Folds -> Domains -> Structure
What is a Fold?
the order and topology of secondary structure elements in a domain
can contain one or more motifs
In: Motifs -> Folds -> Domains -> Structure
What are Domains?
a section of a polypeptide that folds independently of the rest
In: Motifs -> Folds -> Domains -> Structure
What is Structure (tertiary)?
the three dimensional conformation of a natively folded polypeptide
made up of one or more domains
What are common motifs?
helix-loop-helix
coiled coil
helix bundle
beta alpha beta unit
hairpin
beta meander
greek key
beta sandwich
Common domain folds:
parallel twisted sheet
beta barrel
alpha/beta barrel
beta helix
Quaternary structure
the association of two or more natively folded polypeptide chains (subunits) into an oligomeric complex
polypeptides can be different but complex has defined stoichiometry
Each molecule has the same ___ fold but overall ___ is different.
Each molecule has the same GLOBIN fold but overall FUNCTION is different.
Globular proteins
usually water soluble, compact
roughly spherical
hydrophobic interior
hydrophilic surface
Fibrous proteins
mechanical support
collagen
tendons
skin
hair
Membrane proteins
transport
signaling
What is collagen?
a structural protein that is a component of skin, bone, tendons, cartilage, and teeth.
What does collagen consist of?
three intertwined helical polypeptide chains that form a superhelical cable
NOT ALPHA HELICES
HELIX IS NOT AN ALPHA HELIX
What do different proteins in the cell do?
they interact with one another
