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Structural Biology > Protein Folding > Flashcards

Protein Folding Flashcards

1
Q

Landscape Theory

A
  • protein folding is mediated by a complex energy landscape directing the unfolded state conformations into the encoded native genome
  • energy surface representing conformational energy states available to a polypeptide
  • horizontal points represent the conformation/torsion angles and the vertical points represent the free energy associated with this
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2
Q

Protein Folding

A

A protein folds via a series of conformational adjustments reducing free energy and entropy to reach the native state

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3
Q

Anfinsen Experiment

A
  • showed the thermodynamic minimum of the free energy is the native state
  • B-mercaptoethanol allowed reformation of proper disulfide bonds that are most stable and not further reduced
  • native ribonuclease was denatured and reduced with urea and B-mercaptoethanol
  • removing the denaturant without adding reductant left a scrambled protein with non native disulfide bonds
  • trace reductant addition caused a conversion into the native state via shuffling of bonds
  • non native bonds are less stable so are broken and reformed until the native bonds form
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4
Q

Protein Stability

A

structured proteins fold into a native state characterised by a well-defined 3D structure that is more stable / lower free energy
- difference in conformational stability of native state given by free energy difference between native/unfolded states

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5
Q

Chemical Stability

A

ability to maintain the chemical structure of the native state

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6
Q

Conformational Stability

A

ability to adopt a well-defined conformation rather than a random coil state

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7
Q

Determinants of Folding

A
  • unfavourable entropy change of folding a flexible polypeptide
  • favorable enthalpy conditions from H bonds/salt bridges
  • favorable entropy change from burying hydrophobic groups in the molecule
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8
Q

Parameters of Folding

A
  • ph/T/P, ionic strength, crowding
  • parameters of the system can affect the free energy and folding
  • demonstrated in extremophilic organism’s enzymes
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9
Q

Compactness

A
  • proteins are compact with a high density
  • native fold is most stable because of the compactness of helices/sheets
  • folding is largely directed by internal residues
    evidence:
  • modification of Lys residues in Rnase A by poly A doesn’t affect folding
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10
Q

Heirarchy

A
  • folding and structures are heirarchal
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11
Q

Adaptability

A
  • structure is adaptive
  • packing of apolar side chains in core is unique
  • mutagenesis of T4 lysozyme reveals great adaptability in that mutations are accommodated without affecting folding
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12
Q

Sequence Versatility

A
  • conservation of sequence can lead to similar folding
  • 20% aa sequence identified between 2 proteins means they have the same overall fold
  • once you change 50% of the sequence, you change the fold
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13
Q

Techniques for Measuring Stability

A

Anything measuring Protein Stability distinguishes between unfolded and folded states

  • absorbance
  • fluorescence
  • CD
  • NMR
  • catalytic activity
  • urea gradient gels
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14
Q

Circular Dichroism

A
  • essentially a spectroscopy giving the molar absorptivity as a function of wavelength
  • measures molar absorption difference of left and right handed circularly polarized light
  • each secondary structure element produces a characteristic spectra
  • proteins are chiral so interaction with left/right polarised light differently
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15
Q

Circularly Polarized Light

A

Direction of electric field vector rotates about its propagation direction
Forms a helix along the vector of propogation (k)

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16
Q

Ellipticity

A

CD reported as gamma, which is an angle describing an ellipse
- the angle defined by the large/small vectors of an ellipse, ie. the two axes of light

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17
Q

Circular Dichroism Formula

A

Reports difference in absorption of left handed vs right handed circularly polarized light
E = El - Er

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18
Q

Cons of CD

A

over-interpreted & lacks resolution at single amino acid residue level

19
Q

Pulsed HD Exchange

A
  • follows time course of individual residues in a folding protein
  • weakly acidic protons exchange with those of water
  • exchange followed by NMR
  • protein denatured so all residues exposed (specifically amide/hydroxyl)
  • folding initiated in stopped flow analysis and pH increased to initiate H exchange
  • peptide nitrogen atoms who haven’t found H bonds will exchange
  • H/D ratio determined by NMR to show the time course to H bond formation at each residue
20
Q

Levinthal Paradox

A

Too many possible conformations for proteins to fold via random search

  • for a 100 amino acid protein with only 3 possible conformations you have 3^100 conformations
  • therefore, proteins search for the native conformation must be non-random
21
Q

2 State Folding Kinetics

A
  • TS theory is a kinetic model used to analyse the 2 state model of folding
  • high energy metastable TS between the D/N states
  • we use this to study folding kinetics
  • fold rate is proportional to the exponential of the negative activation free energy, the free energy difference between the TS and denatured state D
22
Q

FRET

A
  • used in kinetic analysis to probe conformational transitions in single molecular transitions
  • 2 flourophores: one donor and one acceptor
  • irradiate the donor emitting light in the absorbance range of the second flourophores which emits light
  • if the two molecules are too far away, the acceptor will not emit
  • in the folded state the 2 fp become close enough to emit, giving a high FRET efficiency
23
Q

Stopped Flow Analysis

A
  • rapidly mixes denatured protein with buffer dilutant
  • fit results to an exponential curve to calculate the rate constant, giving kinetic data for folding (can be further used in phi value analysis)
24
Q

Phi Value Analysis

A
  • experimental technique studying TS structure using mutational analysis
  • used to study protein domain folding in a 2 state manner
  • essentially a kinetic/equilibrium ratio
  • folding kinetics/ conformational folding stability are compared with those of point mutants to find phi values shows the mutant residues energetic contribution to the folding TS and revealing the degree of native structure around the residue in the TS
25
Q

Phi Value Results

A 
1 = indicates all mutated residues interactions are formed in the TS
0 = indicates the residue isn't involved in TS stabilisation
26
Q

Structural Refinment

A
  • phi value analysis used for TS modelling
  • gives atomic resolution models of both mutants and wild type folding states and TS states
  • allows us to visualise structural elements in the TS that are key to correct folding
27
Q

Phi Value Calculations

A
  • free energy difference between unfolded and transitional state (mutant-wild type) over the free energy difference between the native and unfolded states (N-D)
28
Q

Process of Folding

A
  • most secondary structure present folds quickly in a burst phase
  • driving force of globular protein folding is hydrophobic group collapse in the core (hydrophobic dye ANS binds to hydrophobic groups and emits in nonpolar environment)
  • molten globule is the initial collapsed state of folding, with significant secondary structure yet disordered side chains and fluctuating structure
  • native-like tertiary structure appears during intermediate folding events (subdomains not properly docked and mobile side chains)
  • final folding requires a series of complex motions permitting attainment of the rigid native core packing and H bonding
29
Q

Trigger Factors

A
  • newly synthesixed proteins leave the ribosome via a narrow tunnel in the large subunit
  • nascent chains are sensitive to aggregation as they emerge in an unfolded state
  • ATP independent chaperone + PPIase
  • binds to ribosome L23 unit and covers exit tunnel to prevent protease contact
  • leaves when chain can fold on its own
30
Q

Hsp70 / DnaK

A
  • detects and sequesters protein chains being synthesized
  • if chain is almost functional it gives time to fold
  • as it folds the protein becomes hydrophilic and releases chaperone
  • brings chain into GroEL complex
31
Q

GroEL/GroEs (type 2)

A
  • 2 copies of the same eptomer
  • 7 copies of the same protein
  • cis (extended with lid) and trans (compact) conformations
  • chamber gives infinite dilution and time to fold
  • 3 domains: apical, intermediate, equatorial
  • protein binds to hydrophobic patches and ATP induced conformational change induces unfolding
  • GroES binds same patches to displace protein into chamber
  • with lid on the chamber exposes hydrophilic surface and extends
  • each unit consumes 1 ATP (7 total)
  • hydrolysis is signal for other chamber to recruit protein
  • alternate 2 stroke mechanism
32
Q

Targets of GroEL

A
  • works on subset of E. Coli proteins
  • a/B proteins with a/B barrels stabilised by many long range interactoins
  • chaperone substrates have similar binding motif to GroES to bind (polar and hydrophobic residues)
33
Q

Heat Shock Proteins

A
  • over-expressed in cases of heat and stress damage
  • favors protein renaturation and degradation to prevent misfolding
  • binds to nascent chains to prevent premature folding
  • prevents folding prior to translocation
  • prevents assembly of multiprotein complexes
34
Q

Protein Disulfide Isomerase

A
  • breaks disulfide bonds to help protein to funnel down energy landscape to native bonds
  • shuffles bonds to find thermodynamically most stable pairings
  • helps conformation of covalent patterns in proteins to form correctly
  • burial of bonds prevents further action
  • oxidative folding : oxidation of reduces cysteine resiudes of nascent proteins allows bond formation
  • strong oxidising agent
35
Q

Peptidyl Prolyl Isomerase

A
  • cis-trans prolyl isomerization is rate limiting in folding of many proteins in vitro
  • PPI accelerates cis-trans isomerisation by twisting peptide bond so atoms are no longer planar
  • helps proline find right conformation
  • lacks NH in amide backbone group so you can have cis conformation of NH amide and carbonyl pointing in the same direction
  • lowers energy barrier/TS energy
36
Q

Protein Turnover

A
  • balance between synthesis and degradation
    1. endosome lysosome pathway degrades extracellular/cell surface proteins
    2. ubiquitin proteasome pathways degrades proteins from cytoplasm/nucleus/ER
37
Q

Lysosomal Degradation

A
  • vesicles containing proteolytic enzymes
  • Hsc73 involved in one pathway
  • protein escorted to lysosome by Hsc73
  • recognises receptor protein on lysosome surface for translocation
  • Hsc73 not degraded but protein is
38
Q

Ubiquitin

A
  • small protein used to tag proteins for degradation
  • linked via isopeptide bond to residues 48/62
  • barcode for destruction : can link to different paths
  • polyubiquitin chains in different combinations can signal differently
39
Q

Ubiquination

A
  • C-terminal Glycine 76
  • 3 enzymes (activating, conjugating, ligase)
  • side chain ubiquination
  • via lysine residues in same mechanism
40
Q

Ubiquitin-Proteasome Pathways

A
  • misfolded and damaged proteins are bound by Hsp70/40 and then polyubiquitination and brought to the proteasome for destruction
41
Q

Proteasome

A
  • recognises ubiquitin to bring substrate inside
  • found in nucleus and cytoplasm
  • cylindrical complex containing 4 stacked rings and a central pore
  • each ring has 7 individual proteins
  • core particle contains active sites and caps regulate entry
42
Q

Proteasome Mechanism

A
  • inner 2 rings made of 7 B-units with active sites
  • outer 2 rings made of 7 a-units with gate function
  • a units controlled by binding to cap particles recognising polyubiquination
43
Q

Aggresome-autophagy path

A
  • specialised type of induced autophagy mediating selective clearance of misfolded and aggregated proteins under conditions of proteotoxic stress
  • K63 linked ubiquination, transport to an aggresome that recruits an autophagic membrane and clears the aggresome via autophagy

Structural Biology (10 decks)

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