Structural Biology
Structural biology is a branch of molecular biology, biochemistry, and biophysics
concerned with the molecular structure of biological macromolecules, especially
proteins and nucleic acids, how they acquire the structures they have, and how
alterations in their structures affect their function. This subject is of great
interest to biologists because macromolecules carry out most of the functions
of cells, and because it is only by coiling into specific three-dimensional
shapes that they are able to perform these functions. This architecture, the
"tertiary structure" of molecules, depends in a complicated way on
the molecules' basic composition, or "primary structures." Structural
biology is that branch of life science, which deals with the study of molecular
structure of biological macromolecules like proteins and nucleic acids.
Bio molecules are too small to
see in detail even with the most advanced light microscopes. The methods that
structural biologists use to determine their structures generally involve
measurements on vast numbers of identical molecules at the same time. These
methods include:
- Macromolecular
crystallography,
- NMR,
- Cryo-electron microscopy
(cryo-EM)
- Multiangle light scattering,
- Small angle scattering,
- Ultra fast laser
spectroscopy, and
- Dual Polarisation Interferometry
and circular dichroism.
Most often researchers use them to study the static "native
states" of macromolecules. But variations on these methods are also used
to watch nascent or denatured molecules assume or reassume their native states.
A third approach that structural biologists take to understanding structure is Bioinformatics
to look for patterns among the diverse sequences that give rise to particular
shapes. Researchers often can deduce aspects of the structure of integral
membrane proteins based on the membrane topology predicted by hydrophobicity
analysis. See protein structure prediction.In the past few years it has become
possible for highly accurate physical molecular models to complement the in
silico study of biological structures.
Structural biology seeks to provide a complete and
coherent picture of biological phenomena at the molecular and atomic level. The
goals of structural biology include developing a comprehensive understanding of
the molecular shapes and forms embraced by biological macromolecules and
extending this knowledge to understand how different molecular architectures
are used to perform the chemical reactions that are central to life.
In addition, structural biologists are
interested in understanding related processes such as protein folding, protein
dynamics, molecular modeling, drug design, and computational biology. Central
tools used in this research include X-ray diffraction, NMR, electron
microscopy, other spectroscopes and biophysical methods, protein expression,
bio-physical and bio-organic chemistry, computer science and bioengineering.
Structural research at MIT
includes groups focusing on: modular signaling domains and protein-protein
interactions; coiled-coil structure, function, and design; structure of Z-DNA,
RNA, and protein-nucleic acid complexes; molecular chaperones that fold and
unfold proteins; G-protein mediated signal transduction; and ab initio protein
design.
Structural biology seeks to provide a complete and
coherent picture of biological phenomena at the molecular and atomic level. The
goals of structural biology include developing a comprehensive understanding of
the molecular shapes and forms embraced by biological macromolecules and
extending this knowledge to understand how different molecular architectures
are used to perform the chemical reactions that are central to life.
In addition, structural biologists are
interested in understanding related processes such as protein folding, protein
dynamics, molecular modeling, drug design, and computational biology. Central
tools used in this research include X-ray diffraction, NMR, electron
microscopy, other spectroscopes and biophysical methods, protein expression,
bio-physical and bio-organic chemistry, computer science and bioengineering.
Proteins
Protein, one of
a large group of nitrogen-rich compounds of high molecular weight that are
essential and abundant constituents of living organisms. Proteins make up over
50 per cent of the dry weight of most cells and only plant cells with their
high cellulose content are less than half protein. There are many different
kinds of proteins, including enzymes, hormones, storage proteins such as those
in the eggs of birds and reptiles and in seeds, transport proteins such as
haemoglobin, contractile proteins found in muscle, proteins
involved in blood clotting and immune defence (antibodies), membrane proteins, and
many different types of structural proteins. Despite this overwhelming
diversity all proteins are made in the same general manner, as linear chains of
amino acids. Each
chain of amino acids, which might contain several hundred amino acids in a
precise sequence, folds up into a unique three-dimensional shape in which its atoms (mainly carbon, hydrogen, nitrogen, oxygen, and sulphur) adopt precise locations. The astronomical number of
possible structures that can be generated in this way produces a huge diversity
of properties and functions. From a chemical standpoint, proteins are by far
the most structurally complex and functionally sophisticated molecules known.
The word protein is derived from the Greek
language and means of primary importance. What are proteins? We are protein.
Our hair, our nails, our skin, our blood, our enzymes and hormones are protein;
indeed, our bodies contain some ten thousand to fifty thousand kinds of
protein. But these proteins are constantly being broken down into amino acids
Amino acids, recycled and built a new, even oxidized to some extent to provide
energy (1 gram of protein weighs in at 4 calories, the same as carbohydrate).
In the typical American diet, about 20 percent of the day's total calories come
from protein.
The term “protein” (from the Greek “pre-eminent” or “first”) was first used by
the Swedish chemist Jöns Berzelius in 1838
for the complex organic nitrogen-rich
substance found in the cells of all animals and plants. Over the following
century these were found to be built from 20 different amino acids and to be
linked together in linear polymers, known as
polypeptide chains. For many years proteins were thought to be amorphous
substances with variable compositions. Progress came with the separation of
individual proteins from complex mixtures and the preparation of crystals of pure
proteins, such as haemoglobin and the enzyme urease. In the late 1930s, Linus Pauling and
Robert Corey in the United States used x-ray diffraction (a
technique in which a beam of X-rays is passed through a crystal of the
substance and the scattered rays measured) to determine the structures of amino
acids and peptides. They also
correctly predicted that many amino acid chains would fold into a stable
conformation rather like a spiral staircase, now known as an - (alpha-) helix.
Another milestone occurred in 1955 when the Cambridge scientist Fred Sanger chemically
analysed insulin, a small
protein hormone, and showed that it was built from a defined set of amino acids
linked together in a unique linear sequence. Everyone now accepts that each
kind of protein molecule has its own unique sequence of amino acids.
In 1955 the American Christian Anfinsen reported that the
small protein ribonuclease (an enzyme) could spontaneously refold into an
active form after being "opened up" by harsh chemicals—evidence that
the amino acid sequence of a protein specifies its function. At the same time,
on the other side of the Atlantic, scientists were probing the
three-dimensional structure of protein molecules. In 1960, John Kendrew and Max Perutz used X-ray
diffraction to determine the three-dimensional structures of the
oxygen-carrying molecules myoglobin and haemoglobin. These studies led to the
concept that linear chains of amino acids fold up to give protein molecules
that have always precisely the same structure and function.
The most important function of protein is to build up, keep
up and replace the tissues in your body. Your muscles, your organs, and some of
your hormones are made up mostly of protein.In the diet, proteins are found primarily in meats, poultry, eggs, milk,
rice and beans, although there are some amino acids in vegetables as
well. The body must digest the food and break it down into singular amino
acids. The food protein cannot be absorbed directly, because the food
sources have polypeptides with hundreds, or even thousands of amino acids
joined together in peptide bonds. These must be broken down with enzymes
into the singular amino acids that the body can absorb, and then combined into
the proteins that the body requires. Heat and processing destroy many
amino acids in dietary sources, and not all food sources have all the amino
acids necessary for proper metabolic balance. Vegetarians do not have
certain amino acids in their diets, such as Lysine, which is found in eggs and
poultry products. A deficiency in even one amino acid will cause
biochemical imbalance creating health problems, and the amino acid(s) should be
replaced for good physical and mental health.
Our body is constantly renewing all its cells and components. Some
cells, like our blood cells, are all completely replaced every month. Others,
are replaced, every couple or few months. The end of a year or so has replaced
every Atom in our body replaced, if we are eating a proper diet. Atoms are much
like rechargeable batteries. They store Solar Energy, by shifting electrons to
higher, more energetic orbits. Our bodies release this stored Solar Energy, by
returning electrons to lower more stable orbits. Plants are Nature's primary
battery chargers. They collect the sun's energy and store it in the electron
orbits of atoms that they have collected from the earth and air. Plants work
with possibly more than 28 different atoms to work their magic. At least 40
different atoms have been found in plants, but so far, only 28 have been
identified as being needed by humans.
This erector set of atoms is used to form, different;
sugars, starches, oils, essential oils, amino-acids, enzymes, Coenzymes,
proteins, vitamins, hormones, on and on. Proteins are the primary component of
numerous body tissues. They are the main component of muscle tissue. Protein
helps muscle development, increases strength and improves athletic performance.
Protein also makes antibodies and hemoglobin (responsible for delivering oxygen
to your blood cells).
Just to sustain life, we need daily
infusions of top-quality proteins because our bodies cannot store them (or
their building blocks, the amino acids) the way they store fats. The proteins we
eat may be complete, meaning they contain all the amino acids we need and in
the portions necessary to good health. Or they may be incomplete (lacking one
or more essential amino acids). They are further categorized as simple proteins
(containing nothing more than amino acids) or conjugated (amino acids bonded
with nonprotein molecules). Collagen, a form of connective tissue, is a good
example of a simple protein; hemoglobin, of a conjugated one (it's composed of
amino acids plus a heme [Iron] group). It's important to know, too, that
proteins can complement one another. That when a food lacking a specific
essential amino acid is eaten with another food that supplies the missing amino
acid, the result is a high-quality protein. Three examples of these complementary
proteins: cooked dried beans eaten with rice, a peanut butter sandwich (i.e.,
bread plus peanut butter) and split pea soup accompanied by corn bread. When a
healthy adult (pregnant women excepted) receives and utilizes the amount of
protein he or she needs, that person is said to be in nitrogen equilibrium.
Pregnant women and growing children are in positive nitrogen balance, meaning
their nitrogen intake exceeds that lost (they need it for growth, building of
new tissue). On the other hand, those with illness or injury exhibit negative
nitrogen balance because they excrete more nitrogen than they consume. As a
general rule, the amount of good-quality protein needed to achieve nitrogen
equilibrium is 0.8 grams per day per 2.2 pounds (1 kilogram) of body weight for
adults. A dire shortfall of protein leads to marasmus, a severe form of
protein-calorie malnutrition common among children in developing countries.
What determines the quality, the biological value of protein? The amino-acid
mix; specifically, the number, type and proportion. Egg whites, with a
biological value of 100, contain the right amount of the right amino acids
needed to meet the body's needs. Indeed, they are the standard against which
other proteins are measured. Good Sources of Complete Protein: Eggs, meat,
fish, poultry, milk and other dairy products.
Proteins are abundant in all organisms and are indeed fundamental
to life. The diversity of protein structure underlies the very large range of
their function. Proteins make up about 15% of the mass of the average person.
Protein molecules are essential to us in an enormous variety of different ways.
Much of the fabric of our body is constructed from protein molecules. Muscle,
cartilage, ligaments, skin and hair - these are all mainly protein materials.In
addition to these large-scale structures that hold us together, smaller protein
molecules play a vital role in keeping our body working properly. Haemoglobin,
hormones, antibodies, and enzymes are all examples of these less obvious
proteins.Whether you are a vegetarian or a ’ meat eater’ you must have protein
in your diet. The protein in the food we eat is our main source of the chemical
building blocks we need to build our own protein molecules. Proteins are very complicated molecules. With
20 different amino acids that can be arranged in any order to make a
polypeptide of up to thousands of amino acids long, their potential for variety
is extraordinary. This variety allows proteins to function as exquisitely specific
enzymes that compose a cell's metabolism. An E. coli bacterium, one of
the most simple biological organisms, has over a 1000 different proteins
working at.
The Building Blocks of Protein:
There are nine essential (L-form) amino acids
that your body requires and must be obtained from food (or supplementation),
since the body cannot manufacture them from other amino acids. These are
Histidine, Isoleucine, Leucine, Lysine, Methionin, Phenylalanine, Threonine,
Tryptophan and Valine.
Non Essential Amino Acids
Non-essential amino acids are just as important, but are
categorized as non-essential since they can be made within the body from other
amino acids. This biochemical conversion is called transamination.
Generally Pyridoxal 5'Phosphate (active B6) is required for this conversion,
and other vitamins and minerals may be also necessary depending on the amino
acid. For example Pyridoxal 5'Phosphate and Vitamin C are necessary to
metabolize Lysine into Carnitine. When the body is lacking sufficient
essential amino acids, or there are insufficient co-factors (vitamins)
necessary for transamination, a deficiency of the non-essential amino acid can
also exist. This can create a metabolic imbalance and possible health
problems, since there would not be enough of all the amino acids necessary to
create the many proteins that the body require for growth and function.
The non essential amino acids are: Alanine, Arginine, Asparagin, Aspartic Acid,
Carnitine, Citrulline, Cysteine, Cystine, Gamma-aminobutyric Acid (Gaba),
Glutamic Acid, Glutamine, Glycine, Ornithine, Proline, Serine, Taurine, and Tyrosine.
Some non-essential amino acids in adults are essential in infants and others
become essential when there are conditions in the body that prevent the
biochemical conversion of one amino acid into another. It is necessary to
constantly replace amino acids to nourish the body and repair and regenerate
tissue. Although we obtain amino acids in food, perfect diets and proper
balance are rarely achieved and supplementation is important to keep the body
in good health. Also in times of physical and emotional stress, illness,
injury and surgery the body requires more amino acids that can be obtained in
foods alone. In addition, many conditions and lifestyles may cause an imbalance
of amino acids and proper balance is critical for good health and longevity.
These are peptide-bonded amino acids from
food sources such as whey, casein, milk and eggs. They may have hundreds
or thousands of peptide bonded amino acids, and your body still needs to digest
them and break them down into the singular amino acids that the body can
utilize. Some of them never get absorbed. These predigested
proteins will also have the good characteristics from food sources that may
cause allergic reactions to food sensitive individuals.UPS Pharmaceutical
grade, or the highest quality, pure, crystalline amino acids are the singular
L-form amino acids. The body best utilizes these, since they do not
require digestion and are easily absorbed. The best delivery system is
capsules (not tablets), since heat and pressure, which are used in most
tablets, can destroy amino acids. A protein molecule is made from a long chain of
these amino acids, each linked to its neighbour through a covalent peptide
bond. Proteins are therefore also known as polypeptides. Each type of
protein has a unique sequence of amino acids, exactly the same from one
molecule to the next. Many thousands of different proteins are known, each with
its own particular amino acid sequence. The repeating sequence of atoms along
the core of the polypeptide chain is referred to as the polypeptide backbone.
Proteins have four levels of
configuration. Primary structure, the amino acid sequence, is specified by
genetic information. As the polypeptide chain folds, it forms certain localized
arrangements of adjacent amino acids that constitute secondary structure. The
overall three- dimensional shape that a polypeptide assumes is called the
tertiary structure. A protein that consists of more polypeptide chains (or
subunits) are said to have a quaternary structure.
Peptide Bonds
Amino acids are linked to each
other by peptide bonds. Peptide bonds are formed by the dehydration of the
carboxyl group of one amino acid and the amino group of the next. Because of
the resonance structure of the electron orbitals on the amino and carboxyl
groups, the peptide bond is planar. The dihedral angle between the amino group
and the alpha carbon and the alpha carbon and the carboxyl group are free to
rotate and these angles are referred to as the phi-psi angles. Glycine, with
the smallest side chain, has the most conformational flexibility about the
phi-psi angles. Other amino acids are restricted in their rotation due to
steric hindrance from the side chains. The rotation of the dihedral angles of
side chains about the different bonds, referred to as chi-1, chi-2 etc., are
also restricted for different side chain elements. Proline, which in which the
side chain is linked back to the backbone is the most restricted; only two
conformations are permitted.
Primary Structure:
The
primary structure of a polypeptide is its amino acid sequence. The amino acids
are connected by peptide bonds. Primary structure of polypeptide determines the
higher levels of structural organization. Amino acids are linked by peptide bonds between α-carboxyl
and α-amino groups. When two amino acids residues are linked in the above
manner resulting in peptide bonds, is called a dipeptide. Thus many amino acids
link together forming a polypeptide in a primary protein structure whose
sequence has N terminus and a C terminus. The N terminus is blocked in few
cases by acetyl group. This polypeptide sequence from N to the C terminus is
the primary protein structure and typically ranges from 100-1500 amino acids
long.
Secondary Structure:
Protein
secondary structure includes the regular polypeptide folding patterns such as
helices, sheets, and turns. The most common types of secondary structure are α-
helix and the β- pleated sheets. Both α- helix and the β- pleated sheet
patterns are stabilized by hydrogen bonds between the carbonyl and N-H groups
in the polypeptide’s backbone.In the 1930s and 1940s, Linus Pauling and Robert
Corey determined the X-ray structures of several amino acids and dipeptides in
an effort to elucidate the conformational constraints on a polypeptide chain.
Both α- helix and the β- pleated sheet called regular secondary structures because they are composed of
sequences of residues with repeating f and c values.
The α Helix
Alpha helices are the most well
known element of protein structure, proposed by Pauling and confirmed in the
first structure determined, myoglobin, and alpha-helices have distinctive
patterns of hydrogen bonding and phi-psi angles. They are generally between 5
and 20 residues in length, but some proteins and coiled-coil structures can be
considerably longer. The carboxyl groups of the backbone hydrogen bond to the
amino group of a residue four amino acids distant along the chain. Alpha
helices generally have a pitch of about 3.5 residues per turn, but there are
forms of helices with tighter (3 residues per turn) and longer (4 residues per
turn). Alpha helices can be coiled about them selves in two coil, three coil
and four coil (four helix bundles) conformations. Alpha helices can exist
internal in proteins (generally hydrophobic), on the surface of proteins
(amphipathic) or in membranes (hydrophobic). Alpha helices can span membranes either
singly or in groups.
β Sheets
Beta-strands are an extended form
in which the side chains alternate on either side of the extended chain. The
backbones of beta-strands hydrogen bond with the backbone of an adjacent beta
strand to form a beta-sheet structure. The strands in a beta sheet can be
either parallel or anti-parallel and the hydrogen-bonding pattern is different
between the two forms. Anti-parallel beta stands are often linked by short
loops containing 3-5 residues in highly characteristic conformations. Longer
loops are occasionally found where the loop plays an important role in
substrate binding or an active site. The antigen-combining site of the immunoglobulin
is an important example of this. Beta sheets can be internal to a protein
(largely hydrophobic) or on the surface in which case they are amphipathic,
with every other amino acid side chain alternating between hydrophobic and
hydrophilic nature. The peptide backbone is constrained by steric hindrance,
and hydrogen bonding patterns that limit its torsion angles (phi-psi angles) to
certain limits. Plots of phi versus psi dihedral angles for amino acid residues
are called Ramachandran plots.
They
have a rippled or pleated edge-on appearance and for that reason are sometimes
called “pleated sheets.” Successive side chains of a polypeptide chain in a β
sheet extend to opposite sides of the sheet with a two-residue repeat distance
of 7.0 Å. The distance between adjacent amino acids along a β- strand is fully
extended. The distance between adjacent amino acids along a β stand is
approximately 3.5 Å, In contrast with a distance of 1.5 Å along the α helix.
Antiparallel β-pleated sheets are more stable than parallel β-pleated sheets
because fully collinear hydrogen bonds form.
- If
extended strands are lined up side by side, H-bonds bridge from strand to
strand. Identical or opposed strand alignments make up parallel or
antiparallel beta sheets.
- Antiparallel
beta-sheet is significantly more stable due to the well aligned H-bonds.
The Ramachandran Plot
In a
polypeptide the main chain N-Calpha and Calpha-C bonds relatively are free to
rotate. These rotations are represented by the torsion angles phi and psi,
respectively. G N Ramachandran used computer models of small polypeptides to
systematically vary phi and psi with the objective of finding stable
conformations. For each conformation, the structure was examined for close
contacts between atoms. Atoms were treated as hard spheres with dimensions
corresponding to their van der Waals radii. Therefore, phi and psi angles which
cause spheres to collide correspond to sterically disallowed conformations of
the polypeptide backbone.
In the diagram
above the white areas correspond to conformations where atoms in the
polypeptide come closer than the sum of their van der Waals radi. These regions
are sterically disallowed for all amino acids except glycine which is unique in
that it lacks a side chain. The red regions correspond to conformations where
there are no steric clashes, ie these are the allowed regions namely the
alpha-helical and beta-sheet conformations. The yellow areas show the allowed
regions if slightly shorter van der Waals radi are used in the calculation, ie
the atoms are allowed to come a little closer together. This brings out an
additional region which corresponds to the left-handed alpha-helix. L-amino
acids cannot form extended regions of left-handed helix but occasionally
individual residues adopt this conformation. These residues are usually glycine
but can also be asparagine or aspartate where the side chain forms a hydrogen
bond with the main chain and therefore stabilises this otherwise unfavourable
conformation. The 3(10) helix occurs close to the upper right of the
alpha-helical region and is on the edge of allowed region indicating lower
stability. Disallowed regions generally involve steric hindrance between the
side chain C-beta methylene group and main chain atoms. Glycine has no side
chain and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot. Hence it frequently occurs in turn regions of proteins where
any other residue would be sterically hindered.
Super secondary Structures
Certain
groupings of secondary structural elements, called super secondary
structures or motifs, occur in many unrelated globular proteins:
·
The most common form of
supersecondary structure is the βαβ motif, in which an α helix connects
two parallel strands of a β sheet.
·
Another common
supersecondary structure, the β hairpin motif, consists of antiparallel
strands connected by relatively tight reverse turns.
·
In α-α motif, two
successive antiparallel α helices pack against each other with their axes inclined.
This permits energetically favorable intermeshing of their contacting side
chains. Such associations stabilize the coiled coil conformation of a keratin.
·
Extended β sheets often
roll up to form β barrels.
Motifs
may have functional as well as structural significance. For example, Michael
Rossmann showed that a βαβαβ unit, in which the β strands form a parallel sheet
with helical connections, often acts as a nucleotide binding site. In most
proteins that bind dinucleotides (such as nicotinamide adenine dinucleotide,
NAD11), two such babab units combine to form a motif known as a dinucleotide-binding
fold, or Rossmann fold.
Tertiary Structure
The term tertiary structure refers to the
unique three- dimensional conformations those globular proteins assumes as a
consequence of the interactions between the side chains in their primary
structure. The following types of covalent and non-covalent interaction
stabilize tertiary structure.
· Hydrophobic interaction:
· Electrostatic interactions
· Hydrogen bonds:
· VanderWaal force of
interaction.
· Covalent bond.
The most prominent covalent bonds in tertiary
structure are the disulfide bridges found in many extracellular proteins.
Cysteine has a thiol group (sulfhydryl hroup) i.e., unique among the 20 amino
acids in that it often forms a disulfide bond to another cysteine residue
through the oxidation of their thiol groups. This Disulfide linkage is an
example of covalent interaction.
a) Interpeptide
hydrogen bonds. Maintain secondary structure.
b) Disulfide
bonds between cysteine residues. According to older definitions these would be
included in primary structure as they are covalent bonds. However, primary
structure is nowadays considered to be the sequence of the amino acids. In
practice disulfide bonds help maintain tertiary or quaternary structures. Since
disulfides are easily reduced to -SH groups inside cells, they are of little
use in stabilizing intracellular proteins. They are mostly used for
extracellular proteins which are exposed to air outside cells.
c) Hydrogen
bonds between R-groups, involving e.g. -NH in ring of His, Trp -OH of Ser, Thr,
Tyr
-CONH2 of Asn,
Gln
d) Ionic bonds
(—NH3+ -OOC—) between R-groups of basic and acidic amino acid residues.
Relatively few of the possible ionic interactions occur in practice. This is
because most polar groups are on the surface of the protein and form hydrogen
bonds to the water.
e) Hydrophobic
bonds between R-groups. The most important influence on tertiary and quaternary
structure. Most proteins fold up so that their polar residues are largely
exposed at the surface. The hydrophobic residues are buried inside, away from
the water. The internal structure is thus dictated largely by hydrophobic
interactions. This is the oil drop model of protein structure. (Note that
intrinsic membrane proteins show an inverse conformation). The strength of
hydrophobic bonds increases with temperature, hence many subunit proteins tend
to come apart at low temperatures. Formation of hydrophobic bonds is driven
mostly by entropy. For example, to dissolve a benzene ring in water - ∆G = +4
kcal (∆H = 0, ∆S = -14). Exposed hydrocarbon residues exert an organizing
effect on surrounding water molecules. Self association of hydrocarbon residues
liberates solvent molecules and so the entropy increases.
Posted by:-Indian Biological Sciences and Research Institute, NOIDA
