Biochemistry: Chapter 4: Protein Denaturation and Folding

methods to recognize protein structure

x-ray crystallography, bimolecular NMR

x ray crystallography

purify protein, crystallize, collect data, calculate electron density, fit residues into density

pros and cons of x ray crystallography

pros: no size limits, well established

cons: difficult for membrane proteins, cannot see hydrogens, biologically active?

bimolecular NMR

purify protein, soluble, dissolve, NMR data collected, find distance between atoms, calculate structure

NMR pros and cons

pros: no need to crystallize, see hydrogens

cons: difficult for insoluble proteins, works with small proteins

protein molecules adopt a specific 3 dimensional ------ in an aqueous solution

conformation

structure adopted by a protein in aquatic solution is called the

native fold

the native fold allows it to fulfill a

specific biological function

in order to fold a protein there is a large cost in

entropy

what drives protein folding

hydrophobic interactions

loss of structural integrity of a protein with accompanying loss of activity is called

denaturation

proteins can be denatured by

ph, heat or cold, organic solvents, chaetropic agents

chaotrope

substance that disrupts three dimensional structure of macromolecules , interferes with inter and intra molecular interactions

ex. urea or guandinium chloride

for complete denaturation also need to

reduce disulfide bonds with beta mercaptoethanol

8M of urea in the presence of beta M fully ----- ribonuclease

denatures

when urea and Beta M are removed slowly the protein--------- refolds and the correct disulfide bonds are reformed

disulfide

the ----- needs to be removed first because of the

urea, covalent bonds forming before the disulfide bonds reform

information present in the ----- sequence is ---- to uniquely specify the correct --- and --- structure of the protein

primary, sufficient, secondary, tertiary

what do hydrophobic interactions promote

aggregation of hydrophobic R groups

what are hydrophobic interactions driven by

the increase in entropy of water molecules surrounding the protein

hydrogen bonds exist largely between the N-H and C double bonded to O groups within the

peptide bond

hydrogen bonds provide precise conformation the

secondary and tertiary structures

vanderwaals interactions can exist between all atoms of a protein but that they are

very distance sensitive

vanderwaals interactions contribute to the

interior stability of the protein

electrostatic interactions (salt bridges) facilitate

long range interactions between charge groups

two theories of the folding pathway for a large polypeptide include

either a hierarchical process or hydrophobic collapse

theory one involves hierarchical process

local secondary structures form first, followed by longer range interaction of those secondary structures

theory two is driven by hydrophobic collapse

which is spontaneous collapse of polypeptide into a molten globule, mediated by hydrophobic interactions, many secondary structures already formed but backbone and side chains are not fixed entirely

most proteins fold by a process that incorporates features of

both models

proteins fold

in the microsecond to second time scales

Levinthal's paradox

finding the native folded state of a protein by a random search among all possible configurations can take an enormously long time

the native structure is the most

thermodynamically stable, lowest energy state

folding is driven by

the search for the most favorable conformation

to direct folding into the correct secondary and tertiary structures no outside agents are

required

Chaperons (Hsp70)

accessory proteins that aid in folding when things go wrong,

chaperons bind and release ---- folded proteins

partially

chaperons also prevent

aggregation or wrong folding

PDI

facilities arrangement of disulfide bonds

PPI

isomerization of peptide bond (cis or trans)

the Tm of collagen from different species is usually just ---- than the ambient temperature of the organism

greater

studies have shown that the ------ of natural proteins can be ----- by the addition of disulfides or by increased packing of internal residues

stability, increased

protein ----- is important for function

flexibility

folding intermediates cannot be --- ---- or proteins will not reach native state

too stable

some proteins must be ---- to cross membranes (mitochondria)

unfolded

some proteins must be ----- for development (collagen in tail)

degraded

diseases that arise from --- of a soluble protein that is normally secreted from the cell

misfolding

misfiled protein is converted into an ------ extracellular amyloid fiber

insoluble

when a protein is misfolded it

induces misfolding in its neighbors

prion protein is produced ---- in neural cells attached to the surface of the cell membrane

normally

cellular and scrappie prions have different

secondary and tertiary structures leading to chronic wasting disease, mad cow, etc.