Lecture 5-7: Protein Folding Flashcards
Non-covalent interactions
Short range repulsion, van der waals interactions, hydrogen bonds, electrostatic forces (ion pairs and salt bridges)
Hydrophobic interactions
Exclusion of water plays role in the folding and stability of proteins
Short range repulsion
2 like charges repel each other when too close and have no attraction when far apart—-affects proteins folding
Van der waals interactions
interacting dipoles from distinct molecules in proteins. Transient.
Hydrogen bonds
Weaker than covalent, but longer than covalent bonds. FON can bond w/ H.
Ion pairs/ salt bridge
This forms a stability for tertiary structure. Ions interacting to cancel out charge.
Hydrophobic effect is
when 2 nonpolar substances come together in solvent(water) and cause water to be excluded from interior of the 2 nonpolar molecules
Secondary structure
A helix (more flexible), B sheet (more rigid and structured). Also, omega loops can be added to give flexibility to B sheets
Bovine insulin
Has both interchain and intrachain disulfide interactions that comprise tertiary structure.
Disulfide bonds Intracellular or extracellular?
Extracellular, the cytoplasm is a reducing environment
Folding funnel timescale
1-rapid formation of 2 structure
2-formation of domains through cooperative aggregation(folding nuclei)
3-Formation of assembled domains (molten globule)
4-Adjustment of conformation
5-More rigid structure
Calmodulin
A Ca++ senior that contains 4 similar A helices, in a single polypeptide. Each unit binds a Ca++ ion
Alternative conformations
Context-dependent; certain protein sequences can be B sheet or A helix based on context of protein location.
Ex of alternative conformation
Lymphotactin; Chemokine structure or glycosaminoglycan binding structure
Determinants of protein folding
2 structure allows for efficient packing, folding is hierarchal (folding funnel), hydrophobic effects, context-dependent
Molten globule state
An intermediate conformational states btw native and fully folded states of a globular protein.
Characteristics of molten globule state:
1-presence of native-like content of 2 struture
2-absence of a tertiary structure (produced by packing of AA side chains
3-Compactness in overall shape of protein, w/ radius 10-30% larger than that of the native state.
4-presence of loosely packed hydrophobic core that increases the hydrophobic SA accessible to solvent.
Molten globule is a compact globule w/ “molten” side chain structure that is primarily stabilized by:
Nonspecific hydrophobic interactions
Protein folding stability is governed by:
non-covalent interactions and hydrophobic interactions
T/F: protein folding is a cooperative process
TRUE
DO proteins prefer higher or lower energy states?
Lower
Molten globule state is
btw native and fully folded state of globular protein
PRimary protein structure
AA linked by peptide bonds
2 structures
Polypeptide chains that can fold into: A helices, B sheets, omega loops (B turns, hairpin turns)
Protein 2 structure stabilizing factors:
Short range repulsion,
H bonds,
van der waals ,
ion pairs and salt bridges
Tertiary structure
WAter soluble proteins fold into compact structures w/ nonpolar cores
Tertiary structure stabilizing factors:
Disulfide bonds, hydrophobic properties
Quaternary structure:
Polypeptide chains can assemble into multisubunit structures
What determines 3D structure of proteins?
AA sequence
How is protein folding/unfolding cooperative?
It is an “All or none” process. Molten globule states is very short. Partial loss of folding / partial fold of structure destabilizes / stabilizes remainder of protein, Structural properties of proteins provide a clear rationale for cooperative process.
Chou-Fasman Method
AA have different propensities for forming secondary structures…the rates of these proteins can be used to predict the 2 structure of the AA sequnce. The higher the number = the higher the P the AA will be in that 2 structure.
Conditions that denature proteins:
Heat, pH (extremes), Agitation
Chemicals that denature proteins:
Detergents (SDS), Chaotropic agents (urea,guanidine hydrochloride). Organic solvents (TCA)
MEthods of analysis of Protein denaturation:
- -Turbidity (light diffraction),
- -Circular dichromism (CD-similar to turbidity but working w/ R/L hand polarized light absorption),
- -UV absorption,
- -Flourescence, biological activity(receptor binding-antibodies)
Circular dichromism
CD- similar working w/ R and L hand polarized light resulting in molecular asymmetry involving a chromophore group.This is used to study the conformation of proteins in sol’n.
EX of Chaotropic agent:
Urea and guanidinium chloride
REducing agents:
Urea, B mercaptoethanol (w/ urea: can be used to reduce ribonuclease), guanidinum chloride
Accessory proteins:
1-PDI (protein DISULFIDE isomerases)
2-PPI (peptidyl prolyl cis-trans isomerases)
3-molecular chaperones (HSP70 AND 90
Protein disulfide isomerases and peptidyl prolyl cis-trans isomerases (PDI and PPI)
Helps proteins to be in proper folding state:
- PDI: rearranges(corrects) non-native disulfide bonds
- PPI: reverses wrong cis-trans formations
Chaperons:
ATP-driven; functions to reverse misfolds, newly synthesizes proteins, unfold/refold of trafficked proteins.
T/F: mitochondria contain their own chaperons?
True; HSP 60 and HSP70…they re distinct from the chaperones in the cytosol.
Molecular Chaperons:
Essential proteins that bind to unfolded and partially folded polypeptide chains, they prevent the improper asso. Of exposed hydrophobic segments, non-native folding, polypeptide aggregation and precipitation will not occur, they allow misfolds do proteins to refold into their native conformations.
2 major classes of chaperons:
1-HSP70: prevent premature folding
2-Chaperonins: w/ large multiple subunit proteins: HSP60 [GroEL] and HSP10 (GroES-cochaperone)
HSP10 and HSP 60 are found in:
Mito. Proteostasis network
HSP70 and 90 is found in :
Cytosol
GroES and GroEL:
HSP10(ES) and HSP60 (GroEL)
MEchanism of HSP10and ;60 (GroES and EL)
1-Unfolded polypeptide enters the cylinder (GroEL-60) from one end
2-The cap (GroES -10) attaches causing the cylinder to change shape;creating a hydrophilic environment for folding
3-Cap comes off, properly folded protein is released.
MEtallopchaperones:
Insert “correct” metal ion into some metal-containing proteins.
Heavy metals (Cd,Hg,and Pb)
Are potent INHIBITORS of protein folding
Common Transport and storage metalloproteins:
Fe and Cu(blue-copper)
Common enzyme metalloproteins:
Mg, Zn, Cu, Fe, Mo, Ni, Co(VIT b12), Mn
Structural difference of aggregated proteins vs. normal proteins
Norm: more A helix and little B strands
Aggregated: More B strands (extended parallel B sheet) b/c they’re sticky. They link together to form amyloid forms.
How do we know structures of aggregated proteins?
Deduced from NMR studies
Quality control mechanisms for misfolded proteins:
Proteosome system and check system
Accumulation misfolded proteins is d.t what 2 things?
Overwhelmingly worked proteosome system and malfunctioning check-system
T/F: Proteins are prone to inappropriate interaction w/ other molecules w/in the crowded environment of a cell.
True
Initiation of amyloid fibrillation:
- seeded polymerization
- covalent modification of proteins: (oxidative, phosphorylation, SUMOylation(small ubiquinated modifiers), proteolytic cleavage modification
Hypothesis of how aggregates lead to cell death:
Smaller aggregates are toxic (not larger ones) d/t cell membrane damage and compromised integrity of the cell.
Amyloid fibrils is derived from:
Amyloid precursor protein (APP)
Amyloid fiber structure:
“Cross beta” w/ B strands perpendicular to the backbone structure
Amyloid formation is characterized by:
Log phase followed by period of rapid growth- this is similar to behavior of nucleated later processes [nucleation]
Protein aggregation is characterized by:
Conformational conversion of soluble proteins into insoluble proteins
Where are amyloid aggregates deposited?
Brain, heart, spleen, liver
Progressing from amyloid to amyloid plaques:
1- seeding [discs stacking]
2-fibril formation [multiple stacks of discs together]
3-deposit
Characteristics of infectious proteins:
Similar in size to virus..poses as infectious character to be passed from cell to cell
3 characters::
1-aggregates of specific proteins
2-resistant to dissolving
3-completely the derived from a cellular protein.
PrP
Prion
Amyloid plaques
B-amyloid
Alpha-synuclein
Plaques
SOD1
Monomer SOD1 plaques
