Thursday, May 22, 2014


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.
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

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