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Protein structure
• Primary structure
• Secondary structure
• Tertiary structure
Primary structure of polypeptides
The primary structure of a protein is specified by the nucleotide sequence of
the corresponding gene.
Consist of amino acids linked together by amide bonds, forming a linear
sequence of 100-1000 amino acids.
Contains all the information to confer both the three-dimensional structure of
proteins in general and the catalytic activity of enzymes i particular.
Secondary structure found in proteins
Secondary structure is a term given to local regions (10-20 amino
acids) of stable ordered three-dimensional structures held together by
hydrogenbonding, that is non-covalent between acidic hydrogens (OH, N-H) and lone pairs.
Some commonly observed secondary structure in proteins is :-helix,
-sheet and -turn.
The -helix is a helical structure formed by a polypeptide chain in which
hydrogen bonds are formed between the carbonyl oxygen of one amide linkage
and N-H of the amide linkage four residues ahead in the chain.
The -sheet is af structure formed by two or more linear polypeptide strands,
held together by a series of interstrand hydrogen bonds.
There are two types of -sheet structures : parallel -sheets, in which the
peptide strands both proceed in the same amino-to-carbibyl direction; and antiparallel, in which the peptide strands proceed in opposite directions.
The -turn is often formed at the end of a -sheet which leads to a 180º turn
in the direction of the polypeptide chain.
Tertiary structure of proteins
The three-dimensional structure of protein sub-units is known as the
tertiary structure. It arises from packing together elements of secondary
structure to form a stable global confirmation.
This involves burying hydrophobic amino acid side chain on the inside
of the protein and positioning hydrophilic amino acid side chains on the
Some common families of protein structure is : -helical, / structure
and anti-parallel  structures
The -helical proteins are made up of only -helices which is pack
onto one another to form the tertiary sturcture.
Structure of cytochrome b562
/ structure
The / structure consist of regular arrays of -sheet- -helix-parallel -sheet
Structure of flavodoxin
Anti-parallel  structures
The anti-parallel  structures consist of regular arrays of -sheet- -turn-antiparallel -sheet.
Structure of suoeroxide dismutase
Watching proteins fold one
molecule at a time
Elizabeth Roades, Eugene Gussakovsky, and Gilad Haran
The article provide evidence for a heterogeneous folding pathway (from
primary structure to fully folded protein) for the protein adenylate
kinase(adenylate kinase catalyse AMP+ATP⇌2ADP). Which is equal to a
rugged energy landscape. It is predicted that a protein on such a surface may
fold via a number of avaible pathways.
One of the major challenges to single-molecule protein-folding studies, is to
restrict the molecule spatially such that temporal folding trajectories can be
measured without modifying the conformtional dynamics of the protein.This
problem is solves by a method where used the folding is followed, this
involves trapping a single protein molecule (mutant C77A of AK from E.coli,
where cystein residues were inserted at position 73 and 203) in a surfacetethered unilamellar lipid vesicles, with a diameter of 120nm. The AK
molecule was labeled at position 73 (acceptor) and 203 (donor) by with
Confirmation of unhindered movement of AK
within the vesicles
Before measuring the folding, its was confirm that AK molecules immobilized
within lpid vesicles, were not interacting significantly with vesicular walls and
thus able to freely diffuse and orient inside the vesicles. This was done by
measuring the distribution o fluorescence polarization values of single vesicleencapsulated AK molecules labeled with donor fluorophore only after excitation
with circularly polarized light. A very narrow polarisation distribution was
measured, which indicate a unhidered rotational motion of the molecules within
the vescles. This is also confirmed by the absent of slow polarization changes.
FRET distribution of AK in vesicles under
native and midtransition condition
The conformational fluctuations of AK molecules, were studie by the use of
fluorescence resonance energy transfer (FRET) between two specifically attched
At first the EET distribution of vesicle-encapsulated labeled AK molecules were
measured under native and denaturing conditions (2M Gdn·HCl, guanidide
hydrochloride). The average FRET efficiency were 0,8 and 0,14 respectively,
which are close to the ensemble values. This means that the encapsulation does not
have an effect on the FRET values.
Because AK molecules are believe to deviate from a two-state folding,
FRET distribution were also measured at 0,4-0,6M Gdn·HCl, which is
around the
midtransition conditions for AK 0,55M Gdn·HCl.
Only the FRET distribution obtained from all trajectories taken at 0,4M
Gdn·HCl is given in the article. This dstribution shows that the
conformational space of the molecules can roughly be divide into two groups
: the denatured ensemle ( EET< 0,45) and the folded ( EET > 0,45).
Examples of single molecule temporal
There are some variability in the behavior of the trajectories. A molecule may
move from one confirmation to another within the same ensemble (e.g B several
transition within the folded ensemble) or transfer from one ensemble to the other
(e.g D transition from the folded ensemble to the denatured ensemble).
Observation of Heterogeneous folding
After time-traces of individual vesicle-trapped AK molecules under
midtransition condition, the EET trajectories were calculated.
From these measurements a map of folding /unfolding transitions were obtained
from singlemolecule trajectories. Each point represents the final vs. Initial
FRET efficiency. Above the line are folding transition and below are unfolding
This shows a very large spread of the transitions. Which indicate strongly a
large heterogenity of the folding reaction of AK.
A historam of the transition step size shows to peaks, one for folding and one
for unfolding. The histogram also show that there is a preference for steps
that change EET by 0,2-0,3.
Thus AK molecules do not typically changes from fully folded to fully
unfolded conformation and v.s in one step. This would require a change in
EET  0,6. Instead AK molecules tend to jump through severeal intermediate
The exact sequence of intermediates changes from one molecule to another.
Which indicate that AK molecules takes a different folding / unfolding
Slow transition
Some trajectories show slow transitions either in the folding or unfolding
direction, many of which take more than a second to complete.
The slow gradual change in EET is a indication of a directed motion on the energy
landscape, possible slow down by local traps. The directed motion can refelct a
highly correlated non-markovian chain dynamics? or a result of slow growth of a
folding nucleus?. Interestly, the slow transitions is not preceded by a fast jump,
which means a abrupt change in EET , this suggest that the barriers sampled by these
molecules are entropic rather then enthalpic.
Other possibilities for signal heterogeneity
•Interaction of the partially denatured protein with the vesicle walls. This was
ruled out by measurement of fluorescence polarization, which confirmed that the
AK molecules were moving freely within the vesicles.
•Jump of the fluorescent labels, thereby modifyng the FRET efficiency. This is
not the case because NO FRET transitions are seen under native conditions,
where orientational jumps should be just as abundant.
•Occurence of cis-proline residues in the native state. Single proline
isomerization cannot explaine the evidence of the large spread in folding
transitions and small-step transitions.
•AK molecules have a preference for small, partial folding/unfolding jumps,
which indicate abundance of local traps on the free energy surface.
•Occurence of slow, directed transitions, which involve correlated motions of
protein segments