Protein chains are like long noodles that are capable of flopping about in different shapes, and of rotating about their axes at many different points (along their length); they can do this because 2 out of every 3 chemical bonds in their backbones allow for rotation of flanking atom groups. Anyone would agree that a noodle tossed repeatedly onto a flat surface (like a table) would be unlikely to fall down in much the same way in any two throws; rather, every throw of the noodle could be expected to generate a different 2-dimensional shape. By extension, a protein chain undergoing folding into a compact 3-dimensional shape, in aqueous solution, would also be expected to adopt a different shape after each attempt at folding. A population of many identical chains protein chains undergoing folding simultaneously could thus be expected to adopt as many shapes as there are chains. Mysteriously, however, this does not happen. Every naturally-occurring protein chain of a defined chemical composition (known as its ‘amino acid sequence’) is found to somehow manage to fold into the exact same three-dimensional structure, suggesting that the amino acid sequence somehow dictates formation of the defined (designate/destined) native 3-dimensional structure. Those who study folding try to understand how this happens…...They examine whether the native structure is adopted through random chain search for a stable structure, or through one or more defined pathways of intermediate compaction steps (also dictated by the amino acid sequence) involving different sections of the chain…. They also examine whether the native structure is necessarily the most stable of all feasible structures. In addition to all this, we also study the role of amino acid nature versus identity, as well as the role of backbone polarity, and the nature of the encoding of folding information into local, or global, elements of amino acid sequence. Protein folding lies at the heart of all living processes. Understanding how it occurs is an issue of the most urgent practical significance. If proteins were not capable of folding, physical life – at least as we know it – could not exist, because proteins are the workhorses of physical living cells. If we understand how proteins fold, we shall someday also be able to understand how and why they misfold, as well as improve them and design/redesign them to do other, newer tasks – not necessarily within organisms, but in chemical reactors and test tubes. We may also be able to design new proteins that help to fight disease, within living organisms.

Generally, of course, proteins fold. However, sometimes they also misfold, and associate into soluble, or insoluble clumps (aggregates). Misfolding can occur because a protein hasn’t had the required physical or chemical environment for undergoing folding, or because it lacked the necessary isolation at critical aggregation-prone stages of folding. Crowding of molecules during folding, or even after folding, is the most common cause of aggregation. However, proteins can also be predisposed to aggregate by mutations in their chemical structures (i.e., changes in the sequence of amino acids that constitute their chain/backbone) occurring at the level of the encoding gene (DNA). Aggregates are significant for several reasons and, therefore, important subjects of study. Below are five of the most important reasons. (1) Aggregates cause diseases. Protein aggregation has been linked to more than fifty human diseases; including some very serious ones like Alzheimers’ disease, Huntington’s disease, and new variant CJD. (2) Aggregates reduce protein yields in biotechnological processes. Many proteins are now produced in microbial cellular factories, including safe bacteria and yeast. Aggregation reduces protein yields from such factories. (3) Aggregates can act as conductors of electricity, and also as proteases. Our group has discovered that aggregates can conduct electricity. Thus, they can affect tissues such as the heart, and brain, and affect normal electrical activity. A collaborating group has discovered that the surfaces of aggregates support a novel proteolytic activity, which might explain the cytotoxicity of aggregates. (4) Aggregates can fluoresce and act as photosensitizing agents. Our group has discovered that protein aggregates display a visible blue fluorescence upon ultraviolet-A illumination. This can photosensitize substances like flavins, to generate free oxygen radicals. (5) Aggregates can be immunogenic, and cause auto-immune reactions. Our group has proposed, and a collaborating group has confirmed, that aggregates can display novel conformational epitopes upon their surfaces. Thus, sites of protein aggregation and deposition in an organism can attract auto-immune attack. Furthermore, many fundamental issues remain incompletely resolved, such as e.g., the issue of whether some of a protein’s normal structure can be retained within aggregates, or whether two proteins of different chemical composition can co-aggregate. The study of protein aggregates has thus grown to be a very important sub-field of the study of protein folding.

Protein stability is intimately linked to protein chain folding/unfolding, and also to the propensity towards aggregation. A protein with a poorly stable three-dimensional structure may be considered to be more likely to undergo partial, or complete, chain unfolding as a part of its normal structural dynamics in an equilibrium situation. In a partially, or completely, unfolded state a protein is vulnerable to undergoing aggregation through collisions with other protein chains in a similar state. Thus, stability is of immense importance in the attainment and survival of a protein’s structure (and function), because even a correctly attained structure would not survive if it were not stable enough. The protein would aggregate, become degraded, or both. Stability can be thought of in two ways. From an equilibrium perspective, thermodynamic stability is important because it determines whether the native structure/state of the protein would be suitably populated (or even populated at all), irrespective of whether the native structure/state has the absolute lowest energy. From a temporal perspective, kinetic stability is important because it determines the rate at which equilibrium is attained. A protein with moderate thermodynamic stability can still be extremely stable from an operational viewpoint if it is highly kinetically stable, with unfolding occurring very slowly, e.g., over a few years, of time, rather than in minutes. Proteins from organisms that grow at high temperatures appear to have evolved such extreme kinetic stability, in addition to a moderately high thermodynamic stability. Understanding the principles by which nature designs/redesigns a protein’s stability – together with understanding the principles of chain folding - is important, for man to be able to build better proteins, as novel reagents capable of working under ‘designer’ conditions.

By definition, engineering activity is related to the manipulation of an object or a process, based on some understanding of that object or process. Man understands some things about proteins today (though, admittedly, not a whole lot). And so, man tries and engineers proteins. Chemically, protein engineering is achieved through organic chemical modifications of the side chains of amino acid residues. Sometimes this involves the addition of a sugar group substitute like polyethylene glycol, to decrease a protein’s immunogenicity, or to increase its survival in a living system. At other times, this involves addition of a reporter chemical group, such as a fluorescent substance which is useful for study of protein behavior, or for diagnostic purposes. Genetically, protein engineering is achieved through designer DNA mutations designed to delete, insert or substitute a particular amino acid, or a group of amino acids (located next to each other on the chain, or far even apart in the chain but close together in the folded 3-D structure). Engineered proteins can have very similar structures to their parent proteins if the level of changes made is small. On the other hand, sometimes a very small change induces a very large effect. At other times, despite a large number of changes being introduced, proteins behave well regardless of whether or not there is a subtle, or profound, change in the structure. Engineering is done with the intention of altering protein structure, stability, and function, and also to understand how folding, and other phenomena occur. We use protein engineering in all these modes, and with all these intents.

Proteins perform binding, sequestration, transport, and catalytic functions. Sometimes, more than one of these functions is found within the same protein, or within a sub-section of a protein known as a structural domain. Protein design/redesign is related to protein engineering, in that it uses the same tools and principles of protein engineering. By definition, when one engineers something there is also an element of design in such engineering design (which is probably why there are courses called ‘Engineering Design’ in many college courses). What distinguishes protein design/redesign from protein engineering is basically ‘scale’. In design, one start as far as possible from scratch, and builds up an entire edifice. In engineering, one starts from an existing natural scaffold, and rebuilds some parts. However, in between these two extremes lie many instances of protein engineering that would also classify to be called protein design, and vice-versa. Basically, given the little that we really understand about proteins, and about their folding and stability, even today, to claim to be involved in ‘protein design’ simply massages a scientist’s ego a little more than to claim to be involved in ‘protein engineering’, although most would probably admit that both terms are misnomers. With some success, man is today able to design/redesign proteins, and engineer them, but the guarantee of success that engineering design requires is simply non-existent. Mainly, it is the successes that get reported. The simple failures lie at the bottom of test-tubes as intractable aggregates. The abject failures do not even get made; they are simply broken down by the cellular factories in which one tries to make them.

Spectroscopy is the science of allowing light to interact with matter, and of inferring things about matter from the nature of this interaction. Sometimes, light is absorbed and emitted as heat. Sometimes, light is absorbed and emitted as light of another (longer) wavelength, in the form of fluorescence or phosphorescence. Sometimes, the absorption itself is distinguished by the structure and geometry of the material doing the absorbing. At other times, light is simply scattered by a system, at the same wavelength or at a different wavelength. The reflectance of light from a surface, following absorption too provides information about a material. The light that is used does not have to be visible. It can be in any of the other regions of electromagnetic radiation accessible to instrumental detection, such as X-rays, radio, ultraviolet, or infrared. The thing within the material that absorbs the light, or reacts to the light, can be an atom, or a set of bonds within a molecule, and the interaction can involve changes in the energy states, or spin states, of electrons or protons, or even other nuclei, or even vibrational states of bonds and molecules. UV-visible absorption spectroscopy allows protein quantitation, and some assessment of purity. Steady-state fluorescence spectroscopy and circular dichroism (differential absorption of circularly polarized light) provides much information about the structural states of proteins, in combination with variations in the use of unpolarized and plane-polarized light. Together with time-resolved fluorescence spectroscopy involving burst-lasers, fluorescence allows protein scientists to even ‘look into’ molecules by allowing assessment of intramolecular distances, protein-protein binding events, and the like. Infrared absorption also provides information about states of folding. Light scattering spectroscopy allows estimation of protein diameters. NMR spectroscopy, which involves the absorption of radio waves in an intense magnetic field, is used to make detailed estimates of three-dimensional structures. X-ray diffraction allows direct determination of protein structures at the atomic level. Thus, spectroscopy – a physics tool - is extremely useful in the study of protein molecules.

Computation in biology comes in many forms. In protein science, it comes primarily in the form of databases for the storage and retrieval of amino acid sequence (or DNA sequence) information, or protein structure-related information and, of course, also in the form of software for the visualization and analyses of amino acid sequences and protein structures. The software for protein analyses are varied, and multifarious. They range from programs that help in the solving of protein structures, based on experimental information from X-ray crystallography, or NMR experiments, all the way to programs that help in the analyses of protein structural characteristics (e.g., atom-atom distances, residue-residue contacts etc.), and modeling of protein structures. Much can be done by computation. We use computation a lot; partly by ourselves, and partly in collaboration with other groups, primarily to visualize, plan, and computationally test the effects of designer mutations upon a protein’s structure, or stability. Basic energy minimization analyses of structures, as well as simulations of protein dynamics, are very useful in determining whether a mutation will be tolerated by an already-formed structure (if there were some way of e.g., ‘waving a wand’ and making a mutation in an existing protein structure. However, this is quite distinct from the question of whether a folding route, or pathway, exists to the designate structure. Thus, computational protein structural analyses are extremely useful; they are good for initially testing out the primary feasibility of structural-biochemical ideas, and mutation-based protein engineering. Ultimately, one has to introduce the mutations and see. Sometimes, ideas that are first tested out computationally work experimentally as well (we’ve been particularly fortunate, in this regard); however, we’ve had our fair share of failures too, where something that works in silico does not work experimentally. Still computation, and especially visualization and structural feature assessments, constitute a very good handmaiden of protein science and protein engineering.

Ah, what can one say about recombinant DNA technology ?! There are tomes written about it, available all over the internet ! Recombinant DNA technology is all about handling, excising, mutating, sequencing and cloning pieces of DNA, and either checking for, or improving, the expression of a gene as a protein molecule. Two or three decades of work by scientists who’ve taken pain-staking efforts to develop reagents have given us the gift of easy availability of a large number of DNA-manipulating enzymes, and other reagents. This availability of reagents makes it is a real pleasure to work in the area of protein engineering, and to do things that would not have been possibly only a few years ago. Maybe we should say, however, that it is not always as easy as it seems. There are many specific hurdles that need to be overcome, especially in every stage of a technique called SOE-PCR (splicing by overlap extension through the polymerase chain reaction), which can give a lot of trouble, and require a lot of trouble-shooting. Still, it needs to be said, that today it is possible to express a protein with virtually any amino acid sequence, as a polypeptide chain, through use of some piece of synthetic recombinant DNA, in one or the other organism, or test-tube reaction. When there are problems in getting a gene to produce a protein chain, there are many ways around these that can be explored. The trick is to get the protein to fold. And sometimes, you can bang your head against the wall, and try every trick in the book, and the protein still won’t fold. With engineered proteins, there’s always the solace that a single mutation can knock out a protein’s ability to fold, as established by the well known case of the single mutation in hemoglobin that causes it to misfold, and give rise to the disease known as sickle cell anemia. When a protein does express, and fold, from a piece of recombinant DNA, the power of the technique is fully manifested.

There are two kinds of protein modifications to talk about. One is the kind that nature introduces. One spends a lot of analytical resources, and thought, in figuring these out. The other is the kind that man can introduce. Amino acids have side-chains that retain the ability to react with other chemical groups. Amino acids like cysteine have a sulphydryl group. Others like serine and threonine have hydroxyl groups. Yet others like lysine and arginine have amine groups. And so on. A bit of synthetic organic chemistry; performed, however, in aqueous media; helps to attach chemical moieties to proteins through the side-chains of some of the amino acids mentioned above. Where one does not exist, it is possible to introduce it through recombinant DNA-based mutations. Thus, proteins can be switched on, or off, through modifications, and especially reversible modifications. Proteins can also be tracked through such modifications, e.g., through covalent adduct formation of some amino acid side-chain with a reagent containing a radioactive element, or fluorescent moiety.

Biotechnology is an all-in-one discipline. There is hardly a science or engineering subject that is not included in it. Call it ‘Biomedical and Biomolecular Biotechnology’ and right there you have already included mechanical engineering (in the form of prosthetic devices, and novel biomaterials); chemical engineering (in the form of proteins, and drugs, generated through fermentative processes in bioreactors); electrical and electronic engineering (in the form of novel instruments for diagnostics, detection, and measurement); civil engineering (in the form of environmental science and engineering through the use of microbes, and other flora/fauna, as well as waste-water treatment through microbes, and pollution control techniques that are ‘green’); physics and chemistry (in the form of all the spectroscopic analysis, protein and DNA modifications and mutagenesis, biomolecular separations technologies involving chromatography and electrophoresis); classical biology (in the form of physiology, biochemistry, microbiology and cell and organism developmental biology, and anatomy); mathematics and computer sciences (in the form of statistical analyses, protein structure modeling, reaction modeling, and databases and programs); and astronomy (in the form of exobiology). A detailed description of ‘Protein Biotechnology’ is provided in PDF link leading from the left panel of the home page of this site.

Proteins know how to (and indeed, do) synthesize every category of biomolecule; perform every sort of transport-, binding-, and catalysis-related function; Proteins define the shapes of cells, of organs, and organisms; they give life mobility; and defend it against other invading forms of life. When allowed to do their own thing, they even know how to shape themselves into exquisite structures, perfectly adapted to designated tasks. They also know how to manufacture a molecule called DNA, and use it as an "Erasable, Programmable, Read-Only" device; An EP-ROM that faithfully stores, and plays back too,.. information that's comprehensive; about how each protein would like to get made, using which components, ....at what levels, .... where , .... and when. Much like a digital diary. Eveventually, of course, you can't do without the organizer. It runs your life; it tells you what to do and when, and you are quite lost without it. But you're comfortable enough, to let it run your life. You don't mind when the world thinks that the tail wags the dog (never mind the mixed metaphors). Which is the tail, and which the dog ? As long as nobody figures out who’s calling the shots, what's a little devolution of control between workmates, now and then ? Master and slave, whipmaster and workhorse, DNA and proteins; now one, then the other. O Proteins ! Versatility is thy name !

The purification, handling and analyses of proteins requires a lot of biomolecular separation methods. In chromatography, proteins are separated by being passed through resins and detected through their absorption, or fluorescence. Separation is achieved on the basis of differences in size (gel filtration, also known as gel permeation chromatography), charge (ion exchange chromatography), surface hydrophobicity (hydrophobic interaction chromatography), overall hydrophobicity (reverse phase chromatography), or affinity to a particular substance (imidazole metal affinity chromatography, immuno-affinity chromatography etc.). In electrophoresis, proteins are separated by being passed through a gel made of polyacrylamide and detected through staining with a dye like Coomassie blue, or by staining with silver, or some fluorescent substances. Separation is achieved on the basis of differences in size alone (SDS-polyacrylamide gel electrophoresis), charge alone (isoelectric focussing), size alone and charge alone, done separately in sequence (2-dimentional chromatography), or on the basis of both size, and charge, together (native polyacrylamide gel electrophoresis). The two methods are invaluable, and are probably amongst the most common of methods used in molecular biology and protein science.