Structure of cytochrome ​c

Cyt​c​ was one of the first mammalian proteins subjected to X-ray crystallography, and the
first 4Å resolution structure was obtained from oxidized horse heart Cyt​c​ in the late sixties.
Higher resolution structures of Cyt​c​ from horse and other organisms were subsequently
published allowing a more detailed view of its basic properties. The heme group is covalently
linked to the Cyt​c​ peptide chain through thioether bonds with cysteine residues 14 and 17
(amino acid numbering is based on the mature peptide, which lacks the N-terminal
methionine). The heme iron is in a hexacoordinate configuration with His18 and Met80 as
amino acid ligands. The heme iron-Met80 bond causes the weak 695 nm absorption band in
the spectrum of Cyt​c​ in the oxidized state. The presence of aliphatic and aromatic amino acid
side chains places the heme group in a very hydrophobic environment, which together with
the iron ligands His18 and Met80 was proposed to explain the high redox potential of Cyt​c​ of
about 260 mV in mammals. Of the entire heme surface only 7.5% are solvent-exposed as part
of the frontal edge of the heme group. This is the site used to transfer electrons from ​bc1​
complex to cytochrome ​c​ oxidase (C​c​O), which was confirmed by co-crystallization of Cyt​c
with ​bc1​ ​ complex from yeast and computer prediction of the Cyt​c​-C​cO
​ complex based on the
bovine C​c​O crystal structure.

Function:
Cytochrome c is a water-soluble mitochondrial intermembrane-space protein loosely attached
to the inner mitochondrial membrane. It is approximately 13 kDa. It plays important roles in
two processes: oxidative phosphorylation and apoptosis.

As an electron carrier in oxidative phosphorylation, cytochrome c shuttles four electrons, one

at time, via its heme group from Complex III (cytochrome c reductase) to Complex IV
(cytochrome c oxidase).

As a crucial player in apoptosis (programmed cell death), cytochrome c is released from the
mitochondria to the cytosol where it binds to an adaptor subunit, APAF-1 in the presence of
dATP, leading to activation of caspase 9. Caspase 9 triggers activation of other caspases
which cleave and destroy other proteins. This results in cell death.

Cytochrome c is primarily known for its function in the mitochondria as a key participant in
the life-supporting function of ATP synthesis. However, when a cell receives an apoptotic
stimulus, cytochrome c is released into the cytosol and triggers programmed cell death
through apoptosis. The release of cytochrome c and cytochrome-c-mediated apoptosis are
controlled by multiple layers of regulation, the most prominent players being members of the
B-cell lymphoma protein-2 (BCL2) family. As well as its role in canonical intrinsic
apoptosis, cytochrome c amplifies signals that are generated by other apoptotic pathways and
participates in certain non-apoptotic functions.

Protein folding models:

Models put forward to explain the phenomenon of protein folding from the random
coil state described above to a native structure must predict two important features
of the process for the simple case of small, single-domain proteins - two-state folding
and co-operativity. These are not mutually exclusive, as a high degree of
co-operativity in the folding of small proteins often leads to the observation of
two-state folding. A two-state folding transition is explained simply as an equilibrium
between a single folded conformation, and an unfolded state as described above,
that is, the transition involves only these two states, with no accumulation of stable
intermediates. The reaction co-ordinate of such a process will consist of two energy
minima separated by a single energetic barrier .The folding of many small,
single-domain proteins is known to follow this two-state approximation, but larger
proteins tend to fold via stable intermediates.

The second important feature is that of co-operativity as determined by the

experimental signature of a sigmoidal transition curve . Co-operativity between weak
interactions is an important concept for much of this thesis, and is discussed further
in the following chapters. Protein structures are held together purely by weak
interactions - hydrogen bonds, electrostatic interactions, van der Waal's packing and
hydrophobic interactions. A single weak interaction contributes very little to the
stability of a protein structure, yet the very large number of these interactions act
together co-operatively to impart high stability to the protein fold. Co-operativity in a
folding process demonstrates that the stability of the folded state is greater than the
sum of the energies of the weak interactions involved.

The first model proposed to explain the co-operativity of protein folding transitions
was helix-coil theory.This represented co-operativity between neighbours in a
polypeptide chain and stated that a residue has a higher propensity to be in a helical
conformation if its neighbour in the sequence is also in a helical conformation. Thus,
a helix can propagate quickly and co-operatively once a few adjacent amino acids
adopt a helical conformation in a chance nucleation event. Although obviously
limited, this theory provides a basis for much work into models for the entire protein
folding process.

Subsequently, many models have been proposed to explain the protein folding
problem. The ``framework model'' describes a step-wise mechanism to greatly
narrow the conformational search.This involves a hierarchical assembly whereby
local elements of secondary structure are formed according to the primary sequence,
but independent from tertiary structure. These elements then diffuse until they
collide, whereupon they coalesce to form the tertiary structure. The ``nucleation
model'' suggests that tertiary structure forms as an immediate consequence of the
formation of secondary structure. Nucleation occurs through the formation of native
secondary structure by only a few residues (e.g. a beta-turn, or the first turn of an
alpha-helix), and structure propagates out from this nucleus. The ``hydrophobic
collapse model'' hypothesises that the native protein conformation forms by
rearrangement of a compact collapsed structure. Hydrophobic collapse to form a
molten globule therefore constitutes an early step in the folding pathway. The
framework and hydrophobic collapse models suggest the formation of kinetic
intermediates, whereas the nucleation model does not. Modifications of the
nucleation model whereby a diffuse folding nucleus is formed and consolidated
through the transition state, concomitant with tertiary structure formation leads to the
``nucleation-condensation model'' proposed by Fersht.
It is likely that folding mechanisms vary significantly according to protein size,
stability and structure. The nucleation-condensation model has been supported by
experimental evidence from several small proteins including chymotrysin inhibitor-II
and barstar. Bychkova and Ptitsyn have studied more than 20 proteins and found
that nearly all adopted a molten globule state under mild denaturing conditions. This
points to the hydrophobic collapse model, a model favoured by many for the case of
larger proteins.
A more general description of protein folding pathways has emerged more recently
termed ``folding funnel'' theory. This represents the energy surface of a protein
folding pathway as a funnel, with a diverse multitude of unfolded conformations at
the rim, and a single global minimum representing the native folded conformation.
This leads to a ``new view'' of protein folding whereby many paths exist from the
unfolded to native states, and a protein molecule may follow the steepest (fastest
folding) path, or a more round-about route passing through several local minima
(intermediates) and maxima (transition states).

Levinthal’s Paradox
Energy landscape for the folding of the protein Rhodanese. The landscape on the left shows is the
landscape for folding in solution, while the landscape on the right shows the landscape for folding mediated
by SR1. Note that the lowest energy corresponds to the fully folded state. Figure taken from Reference 2

Definition
It is well known that proteins in solution can reliably fold from a random coil to a unique native
conformation on a biologically relavent timescale. Levinthal's paradox is an apparent
contradiction between the number of possible conformations for a protein chain and the fact that
proteins can fold to their native conformation quickly (less than a second). In the proceedings
where Levinthal first mentioned the paradox that bears his name (Reference 1), he estimates
that for a protein there are 10​300​possible conformations. Using the amount of time it actually takes
for a protein to fold to its native conformation and assuming the minimal amount of time to
sample different conformations, the protein would only be able to sample ~10​8​ different
conformations if the protein sampled the conformation space randomly. Because of the relevant
timescales, if the protein randomly sampled conformation space, a protein would not be able to
correctly fold in a person's lifetime. Herein lies the paradox, since properly folded proteins are
needed for the person to exist in the first place.

Possible Solution
Because of the paradox, it becomes obvious that the protein cannot sample conformation space
in a random fashion. Instead, the protein samples the space in a way consistant with statistical
mechanics. The probability of sampling a given conformation is weighed by the energy of the
conformation. Currently, the generally accepted idea in the field of protein folding is to consider
an energy landscape, where every possible conformation is represented by an energy value. The
idea is that the energy landscape is generally funnel shaped, with the native conformation having
the lowest energy. As the protein folds from its random coil conformation, it travels from
conformation to conformation such that with each step, the total energy is lowered. With the idea
of a funneled energy landscape, it may seem that it would be possible to calculate the energy
landscape of a given amino acid sequence and find the conformation with the lowest energy,
corresponding to the native structure. However, in practice this is not the case and we can only
predict the native structures for relatively small proteins.

Difference Motifs and domain

These are completely different concepts, which sometimes may be connected.

A motif in biology is a mathematical model, typically of a sequence, that predicts which

sequences belong to some defined group. For example, a DNA sequence motif can characterize
the binding site of a transcription factor, i.e. which sequences tend to be bound by this factor. For
proteins, sequence motifs can characterize which proteins (protein sequences) belong to a given
protein family. A simple motif could be, for example, some pattern which is strictly shared by all
members of the group, e.g. WTRXEKXXY (where X stands for any amino acid). There are also
more complex motif models.

Protein domains, on the other hand, are a structural entity, usually meaning a part of the protein
structure which folds and functions independently. So, proteins are often constructed from
different combinations of these domains.
Secondary and tertiary structure: motifs and domains

● ​Motifs​​ (supersecondary structure)

simple combinations of secondary structure

formed from consecutive sequences of primary structure

helix-loop helix (EF hand), b-a-b, Greek key

● ​Domains

stable, independently folded, globular units, often consisting of combinations of motifs

vary from 25 to 300 amino acids, average length – 100.

large globular proteins may consist of several domains linked by stretches of polypeptide.
Separate domain may have distinct functions (eg G3P dehydrogenase). In many cases binding
site formed by cleft between 2 domains

frequently correspond to exon in gene

● ​Some examples of domains:

1. in volving a-helix

4-helix bundle

globin fold

2. parallel b-sheets

hydrophobic residues on both sides, therefore must be buried.

ab barrel: 8 b strands each flanked by an antiparallel a-helix eg triose phosphate isomerase.

eg Fig 4.23 -Horton)

3. antiparallel b-sheet

hydrophobic residues on one side, one side can be exposed to environment, minimum
structure 2 layers

Sheets arranged in a barrel shape

More common than parallel b-barrels

eg. immunoglobulin