Professor Uglade Lecture Flashcards
Non polar hydrophobic amino acids
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Glycine (Gly, G)
Leucine (Leu, L)
Alanine (Ala, A)
Methionine (Met, M)
Phenylalanine (Phe, F)
Valine (Val, V)
Isoleucine (Ile, I)
Proline (Pro, P)
Tryptophan (Trp, W)
Cysteine (Cys, C)
Tyrosine is also hydrophobic due to aromatic ring in side chain (even though its in polar group)
-Cysteine can form disulfulide bonds between 2 cystein side chains in proteins (covalent bonds)
Charged amino acids
Basic (positive charge)
BLAH
L-Lysine (lys, K)
A- Arginine (Arg, R)
H-Histidine (His, H)
ADE
Acidic (negative charge)
D- aspartate (Asp, D)
E-Glutamate (Glu, E)
The charged R groups allow for electrostatic ionic interations between the acid and basic amino acids
what are the Uncharged Polar amino acids, and what bonds do the R groups form
R groups form hydrogen bonds
STaNQY
S-Serine (Ser, S)
T-Threonine (Thr, T)
N-Asparagine (Asn, N)
Q-Glutamine (Gln, Q)
Y- Tyrosine (Tyr, Y)
Tryptophan (Trp, W) can also form H bonds due to NH in side chain (even though its non polar)
how are polypeptides made, what type of rxn is this?
-what do the side chains determine?
-What do side chains and backbone form?
-made by a covalent amide linkage called peptide bond (uncharged but polar)
-condensation reaction
-side chains determine charge and hydrophobicity
-side chain and backbone can form non covalent contacts with other AA
What are the peptide bonds like in the Polypeptide backbone, where is rotation possible?
-peptide bond is planar and cannot rotate
-rotation around central alpha carbon is possible (has some felxibility)
what are the 4 types of non covalent bonds
- hydrogen bonds (polar uncharged amino acids, Asn, Gln, Ser, Thr, Tyr)
- van der waals interactions (all amino acids have this)
- ionic bonds (polar charged amino acids, Asp, Glu, Arg, Lys, His)
- hydrophobic interactions (exclusion of water)(non polar amion acids, Ala, Gly, Val, Leu, Ile, Pro, Phe, Met, Trp, Cys)
covalent interactions
disulfide bonds between 2 cysteines
-cytosolic proteins normally do not have sulfide bonds
4 organization levels of protein structures
- primary structure- linear amino acid sequence
- secondary structure- local conformation patterns
- tertiary structure- proteins acquire function from this conformation
- quaternary structure- assembly of multiple polypetides into a final protein
- amino acids peptide bonds and side chains engage in non covalent interactions to form 2nd, tertiary, and quaternary protein structure
secondary structure
-alpha helix- Results from H bonding in polypeptide backbone between N-H and C=O (formed every 4 peptides, alpha helix turns around every 3.6 amino acids)
- beta sheet- hydrogen bonds between backbone strands, very rigid, 2 types (parallel and antiparallel)
-commonly alpha helices and beta sheets
tertiary structure, what is the arrangement?
What types of contacts are formed?
What are the long range contacts between?
What structure do loops not have, why?
3-D arrangement of the polypeptide
-secondary struture elements are packed against each other to form a tetiary structure
-hydrophobic contacts between 2ndary elements
-long range contacts between residues that are far apart in the primary sequence
-loops have no secondary structure so they can be flexible
quaternary structure
-assembly of multiple polypeptides into a final protein
-interactions between subunits are very stable
-dimer- 2 polypeptide subunits
-trimer, tetramer, 5-mer, etc (oligomers- many subunits)
domains
-independently folded unit within a protein
-proteins can have one or multiple domains
-different domains in a protein often have diff, functions
-conserved in evolution
-forms non-covalent interactions with other molecules
-50-200 amino acids long
-long proteins have multiple domains (most polypeptides 100-800 amino acids in length)
amino acid side chain, what does it dertermine, and interact with
- determines charge, polarity, and hydrophobicity
- and interaction with other molecules (function)
what does sequence similarity indicate?
What does homology indicate?
what happens when polypeptides have diff sequences?
-homology (similarity) indicxates evolutionary conservation
-homology suggests common structure or function
-if polypeptides do not have sequence similarity they are divergent
Post translational modification (PTM) importance
-important for protein diversity, complexity and essential for regulation of protein function and cellular signaling
Types of PTM, what does cleaving, what do we do to side chains and N-terminus?
- cleaved into smaller pieces by petidases
- covalent modification of N-terminus (only modification to occur post translationally for N-terminus)
- covalent modification of side chains: introduce functional groups to proteins
Side chain modifications
What can they change? What can they create?
What are many of the modifications, what are they useful as?
-used for various cell functions
1. can change surface or conformaiton of protein
2. can create or block a binding site for other proteins
3. many mods are regulated and reversible
4.modifications are fast, so useful as switches
-all mods are mediated by enzymes
Main Types of side chain modifications
Phosphorylation (addition of phosphate)
methylation (addition of methyl groups)
acetylation (addition of acetyl group on lysine residue, commonly on N-Terminal)
-glycosylation, sumoylation, ubiquitination
PTM: phosphorylation
30% of proteins get phosphorlyated
-Only 3 amino acids can get phosphorylated on hydroxyl groups (SYT):
-Serine (S), Tyrosine (Y), Theronine (T)
- adding phosphoryl group changes the charge and size
-Kinases and Phosphatases are involved in this process (process is reversible)
what do kinases transfer?
what always particaptes in phosphor?
-what are the 3 types of kinase?
-transfer phosphates from ATP (donates group to serine)
-ATP always participates in phoshorphylation
3 types on kinase families:
-Ser/Thr kinases (adds grou to ser+thr)
-Tyr kinases
-dual specificity (ser/Thr and Tyr)
Example- aspartic acid and phosphoserine are very similar (neg charge, similar size, form ionic bonds)
phosphatases
remove phosphate groups
Phosphatase families:
-Ser/Thr phosphatases (removes group to ser+thr)
-Tyr phoshphatases
-dual specificity (ser/Thr and Tyr)
Example- serine and alanine are similar dephosphorylated
phosphopeptide bonding
-phosphorlyation can be signal to degrade protein, it is required for binding
-specialized domains bind to p_ser, p-Thr, or p-Tyr
example: phosphor of Thr allows for interactions eith other proteins through non covalent bonds
what does Acetylation of lysine change?
-What recognizes specific sequences?
What is acetylation used for, what does it increase?
-changes polarity (removes positive charge, no longer can do ionic interactions)
- Lysine (K) acetyltranferases (KATs) and deactylases (KDACs), recognize specific sequences
-used for signaling and metabolic effects
-increases size and changes charge
Example- lysine is acetylated and interaction with DNA will be weak and chromatin will not be tightly bound which is imp. for regulation of transcription
Methylation of Lysine (K) and Arginine (R)
-addition of 1 or 2 methyl groups to the guanidino group
- adds size to K and R
-methylation does no change charge but it changes the capacity of the protein to interat with others
-lysine can be mono, di, or tri methylated
-Lysine methyltransferases (KMTs) and Lysine demthylases (KDMs)
PTM binding, what are they and what do they provide?
Acetylation and methylation provide new binding sites for proteins like phosphorylation
-certain domains bind Ac-Lys, Me-Lys, Me-Arg and surrounding sequences
what does the folded structure depend on on the interior and exterior surface?
What is most stable conformation?
-folded structure depends on hydrophobic interactions on interior of structure
-polar side chains on outside surface
-native state is completely folded conformation of a protein (lowest state of energy, and is determined by amino acid sequence)
-side chain mods take place after folding usually
important interactions for folding
- secondary structure has lots of H-bonds
-tetiary structure has more hydrophobic interactions based on binding
Is the Folding process complex and how does it affect the structure of the protein (length)?
What do unfolded domains do?
-complex process
-unfolded (denatured) domains have extended conformations with no 2nd, or tertiary structure
-folding increases structure
what is the Native state, what is it stabilized by, what do some domains require?
-most stable conformation of protein
-stabilized by hydrophobic contacts (exclusion of water)
-some domains require ligand partner to be stable (cofactor (haem, steroid, etc) or another protein subunit)
what structure do folding intermediates have?
what is exposed, and what is the state of the polypeptide?
-have some 2nd structure but tertiary incomplete
-some hydrophobic side chains exposed
-more of polypeptide is flexible/disordered
HSF1 regulation
1. What does folded HSF1 mimic/bound to?
2. What do unfolded proteins do after heat shock?
3. what does HSF1 do?
4.why is HSP90 expressed?
5. How is HSF1 downregulated?
- monomeric HSF1 is folded, but mimics unfolded protein and is bound by Hsp90
- after heat shock, unfolded proteins compte with HSF1 for HSP90 binding
3.free HSF1 trimerizes and activates transcription - chaperones including Hsp90 are expressed and help fold or degrade the unfolded proteins
5.HSF1 is downregulated by binding excess Hsp90 to the monomer form
HSP70 functional cycle
1. What delivers substrate to ATP-bound HSP70?
2.What is hydrolysis mediated by, what does this result in?
3. what does NEF do?
4. What is opning of alpha helical lid induced by?
5. what happens to released substrate?
MHNOR
M-Hsp40 mediated delivery of substrate to ATP-bound Hsp70
H-hydrolysis of ATP to ADP mediated by hsp40 resulting in closing of alpha helical lid and tight binding of substrate Hsp70
N-NEF catalyzes exchnage of ADP for ATP
O-opening of alpha helical lid, induced by ATP binding, causes substrate release
R-released substrate either folds to native state or repeats another cycle
HSP90 functional cycle
-When is the substrate bound weakly?
-What does ATP binding allow?
What does ATP hydrolysis do?
- substrate bound weakly in the open nucleotide free state
- ATP binding allows dimer to close and bind substrate tightly
- ATP hydrolysis to ADP compacts the dimer and releases substrate
HSP70-HSP90 co chaperone system, what does HOP assist in?
-When does HSP70 dissociate?
-What do co chaperones provide/act on?
What happens when folding is not complete?
-HOP assists complex formation
-HSP70 dissociates when HSP90 binds ATP
-Many co-chaperones regulate HSP70 and HSP90
-co chaperones sometimes act on substrates
-provide flexibility
-folding and non folding functions
-if folding not complete in first round it goes through 2nd cycle
What do GroEL subunits have?
-What ATPase domain the interface for?
-What is the movement of substrate controlled by?
-Each GroEL subunit has and ATPase domain and substrate binding domain
-ATPase domain is the interface with the opposite ring (upside down)
-movement of the substrate binding domain is controlled by the ATPase in both rings
