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PHYS3522 Bionanophysics 1 > Modelling Proteins > Flashcards

Modelling Proteins Flashcards

1
Q

What type of polymer are proteins?

A
  • linear, random heteropolymers composed of amino acids
  • rather than being a random coil, many proteins have a well defined structure
  • complex interplay between polymer rigidity, chain entropy, monomer-monomer interactions and monomer-solvent interactions
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2
Q

Single Molecule Force Spectroscopy of Proteins

Intramolecular Forces

A
  • polyprotein chain attached to gold substrate at on end and pointed end of cantilever at the other
  • laser bounced off of cantilever to measure the force exerted
  • cantilever pulls on the polyprotein chain causing the domains to unravel one by one
  • in a chain of different domains, the weakest always break first
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3
Q

Single Molecule Force Spectroscopy of Proteins

Intermolecular Forces

A
  • one molecule attached to the substrate
  • other molecule attached to the cantilever
  • lower the cantilever towards the other molecule and measure the force on the cantilever
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4
Q

Other SMFS Techniques

Optical Tweezers

A
  • molecules tethered to a planar surface and beads held by an optical trap
  • distance changes are obtained from the motion of the bead
  • forces obtained from the displacement of the bead from the centre of the trap
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5
Q

Other SMFS Techniques

Magnetic Tweezers

A
  • molecules tethered to a planar surface and magnetic bead held by magnetic trap
  • distance changes obtained from the motion of the bead
  • forces set by the strength of the magnetic field
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6
Q

Mechanical Stability of Beta Sheets

A
  • beta sheet proteins tend to be mechanically strong
  • this is because shearing apart of two beta sheets requires simultaneous rupture of the non-covalent interactions along the length of the sheet
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7
Q

Mechanical Stability of Alpha Helices

A
  • alpha helices tend to be mechanically weaker

- this is because helices can unfold in a step-wise sequential manner

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8
Q

Force Unisotropy

A

-the direction of applied force relative to the topology of the secondary structure determines the mechanical strength of the protein

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9
Q

Mechanical Clamp

A
  • some beta sheet proteins have mechanical clamps
  • a series of hydrogen bonds along the shearing interface
  • it is the breakage of these bonds that is the rate limiting step in protein unfolding
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10
Q

Freely Jointed Chain Model

Define Parameters

A

-polymer is a chain of N perfectly rigid segments of length b, the Kuhn length
-no restriction on the angles between adjacent segments, no energy is required to change the angles
-no interactions between segments (except the end-to-end connectivity)
-contour length:
Lc = bN
-where N is the number of segments

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11
Q

Freely Jointed Chain Model

Forces

A

-tensile force tends to align the segments in the direction of the force
-opposing the stretching is the tendency of the chain to maximise its entropy
-extension corresponds to the equilibrium point between the external force and the entropic elastic force of the chain
-at low forces the force-extension relationship is give by:
F = 3kbT/b * x/Lc
-the FJC describes a Hookian spring relationship with a spring constant kb*T/b between the unfolding force and the extension of a polymer at low force
-the spring constant is proportional to the temperature reflecting the entropic origin of chain elasticity

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12
Q

Freely Jointed Chain Model

Limitations

A
  • the FJC model is good at low and high forces but not in between
  • this is because the FJC model simplifies the description of the polymer molecule:
  • each Kuhn segment has a fixed length, is unstretchable and completely straight
  • -no thermal fluctuations away from the straight line are allowed
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13
Q

Worm-Like Chain Model

Description

A

-the WLC model describes the behaviour of semi-flexible polymers
-the entropic elasticity of the polymer involves small deviations of the molecular axis due to thermal fluctuations
-the direction of the chain is correlated over a distance, the persistence length Lp
-the persistence length Lp quantifies the stiffness of the chain:
cos(β) = e^(-L/Lp)

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14
Q

Worm-Like Chain Model

Forces

A
  • an interpolation formula has been numerically determined to describe the relationship between the applied tensile force, F, and the extension x of the polymer
  • for small forces (and small extensions) the WLC model behaves like a Hookian spring with a linear relationship between force and extension
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15
Q

What can we learn using single molecule force spectroscopy?

A
  • a protein’s mechanical stability is determined by intramolecular interactions and the direction of the applied force
  • we can use polymer models to examine force-extension behaviour
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16
Q

What can we learn using single molecule force spectroscopy?

A
  • a protein’s mechanical stability is determined by intramolecular interactions
  • a protein’s mechanical stability is determined by the direction of the applied force
  • we can use polymer models to examine force-extension behaviour
17
Q

Unfolding and Unbinding

Definitions

A
  • unfolding: disrupting intra-molecular bonds

- unbinding: disrupting inter-molecular bonds

18
Q

Protein Unfolding/Unbinding

Description

A

-protein mechanical unfolding/unbinding are stochastic processes described by a lifetime

19
Q

Effect of Force on Bond Lifetime

A
  • force acts as a denaturant by diminishing activation barriers to unfolding/unbinding
  • an external force tilts the energy landscape by F(x-x0)
20
Q

Single Bond Lifetime Without Force Present

Arrhenius Expression

A

τo = 1/vo * e^(ΔG/kb*T)

-where vo is the characteristic vibrational frequency

21
Q

Unbinding Mechanics of Multiple Bonds

Unfolding

A
  • disrupting intra-molecular bonds
  • two beta sheets hydrogen bonded together
  • force being applied parallel to the clamp in opposite directions at opposite ends of the sheet on each chain
  • bonds in parallel
22
Q

Unbinding Mechanics of Multiple Bonds

Unbinding

A
  • disrupting inter-molecular bonds
  • two beta sheets hydrogen bonded together
  • force applied perpendicular to the sheets at the same end
  • zipper arrangement
23
Q

Unbinding Mechanics of Multiple Bonds

Zipper Arrangement

A

-bonds break in sequence at random times from first to last
-assuming individual bonds break independently, the total lifetime is the sum of the individual bond lifetimes
-for N identical bonds:
τ1 = τo * e^(-FΔxu/kb*T)
-so
τN,zipper = τ1 * N

24
Q

Unbinding Mechanics of Multiple Bonds

Bonds in Parallel

A

-bonds break one by one in random order and the external force is shared amongst the remaining bonds
-give the first bond to break index N and the last bond to break index 1
then, for the ith bond to break, the force is F/I and the lifetime needs to be scaled by 1/I:
τ1 = τo/i * e^(-FΔxu/ikbT)
-so:
τn,parallel =
τo * Σ[ 1/i e^(-FΔxu/kbT)]
= Σ[ 1/i * τ1^(1/I)]
-where the sum is taken from i=1 to i=N

25
Q

Catch Bonds

Definition

A
  • a type of non-covalent bond whose dissociation lifetime increases with the tensile force applied
  • conceptually similar to a chinease finger trap
  • the opposite of what we would normally expect, usually increasing tensile force would decrease bond lifetime
26
Q

Catch Bonds

One-State Two-Path Model

A
  • an alternative dissociation pathway is introduced along which the system can dissociate against low external forces resulting in a tightened bond for larger forces
  • as a certain critical force the system reaches maximum stability and switches to the slip dissociation path
27
Q

Catch Bonds

Two-State Two-Path Model

A
  • two distinct bound states, B1 and B2
  • both states individually obey slip bond characteristic and dissociation can occur from either of the two state depending on the external force
  • the relative occupancy of the two bound states is force dependent
  • at low force the system dissociates directly from state B1
  • at high force the system crosses to B2 and then dissociates
  • this can be achieved through spatially separated bound states or through a force-induced switch in protein molecular conformation from B1 to B2
28
Q

What are three examples of where catch bonds are found?

A
  • cell adhesive molecules
  • intracellular bonds exposed to force
  • bacterial adhesive molecules
29
Q

Protein Folding

Levinthal’s Paradox

A

-Levinthal pointed out that since each amino acid has three accessible conformations, even for a short polypeptide chain of 100 amino acids the protein would have to sample 3^100 conformations in its search for a single native conformation
-since it takes 10^(-13)s for a chemical bond to to rotate it would take 10^27 years to do this
-in reality polypeptide chains fold into their unique 3D structures in a matter of seconds by a random search of the available conformation space
=>
-there must be defined pathways for a protein to old

30
Q

What are the three conceptual models of protein folding?

A
  • framework model
  • nucleation and growth model
  • hydrophobic collapse model
31
Q

Conceptual Models of Protein Folding

Framework Model

A
  • sequential formation of native-like micro domains (alpha helice, beta sheets etc.)
  • these small secondary structural units are formed locally during the initial stage of protein folding and come together by random diffusion and collision
32
Q

Conceptual Models of Protein Folding

Nucleation and Growth Model

A
  • a few key residues of the polypeptide chain form a local nucleus of secondary structure in the rate-limiting step of folding
  • around this nucleus, the whole native structure develops as in a crystallisation growth process
33
Q

Conceptual Models of Protein Folding

Hydrophobic Collapse Model

A
  • folding begins by an initial clustering of hydrophobic residue chains which prefer to be excluded from an aqueous environment
  • the clustering of hydrophobic residues is expected to be non-specific and happen rapidly
  • the formation of an ensemble of collapsed structures would rapidly reduce the available conformational search-space
34
Q

Which is currently the best model for protein folding?

A

-hydrophobic collapse model

35
Q

Energy Landscape Theory

A
  • statistical mechanics based models postulate that protein molecules traverse a funnel-shaped energy landscape during folding
  • and the protein folding pathways more closely resemble funnels than random diffusion in the configuration space
  • a folding funnel is a plot of the free energy against configurational entropy
  • an individual folding trajectory is envisaged for each polypeptide chain traversing down the folding funnel
36
Q

Protein Folding and Force

A

-protein folding can be impeded by force

37
Q

Force on Bonds in a Linear Chain of Protein Domains

A
  • in a chain of N proteins, the force on each bond is still F
  • only one bond has to break for the entire chain to break
  • there are N possible breaking points each with the same breaking probability as a single bond
  • the probability of breakage is therefore N times greater than a single bond

PHYS3522 Bionanophysics 1 (12 decks)

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