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Basic concept for designing a new protein/enzyme molecule As we know that large number of proteins/enzymes found in nature, which fold into a variety of different structures and carry out a huge diversity of functions. Protein engineering attempts to design protein/enzyme structures, including those having particular functions. Therefore targets for enzyme/ protein engineering are · enhancement of enzyme activity, · improved stability of the protein, · altered pH optima or temperature tolerance and · modified specificity Researchers can improve the knowledge we have about the forces and effects that specify the properties of the folded states of a protein. In addition, control over the design of particular folded state structures will likely lead to new synthetic proteins having the efficiency and specificity of biological proteins. Designing a new protein/enzyme molecule has applications include therapeutics, sensors, catalysts, and materials. The successful design of proteins/enzymes is possible even without a complete quantitative understanding of all the forces involved in specifying their structures. Designing of proteins/enzymes is nontrivial, however, because of both their complexity and the delicacy of the interactions that specify the folded state. Proteins are macrobiomolecule and having many structural variables specify the folded state, including sequence, backbone topology, and conformations. There are two main motivations for designing of proteins/enzymes: side-chain The first is based upon the assumption that a complete understanding of any natural system depend upon our ability to design a similar artificial system from first principles. The second motivation is for de novo protein design is one of the practical approaches. Therefore our understanding of natural proteins for their folding pathways, thermodynamic stabilities and catalytic properties is enhanced by our ability to design novel proteins with predetermined structure and properties. The ability to design proteins/enzymes de novo has the potential to bring a revolution in the field of science and technology ranging from industrial catalyst to biomedical engineering. Protein/enzyme design also refers to the effort to design new protein molecules of a desired 3-D structure and function. It is a reverse procedure of protein structure prediction and the solution of the problem therefore highly relies on the extent of our understanding on the principle of protein folding. How ? Fig: Protein design is a reverse procedure of protein structure prediction. Finding out an amino acid sequence that will adopt a unique and stable three dimensional structure is the main goal to design a novel protein. The fundamental hurdle to design a novel protein is the conformational entropy of the linear polymer chain which must be overcome. The conformational entropy represents a substantial amount of unfavorable free energy. For a design to succeed the favorable free energy associated with these designed interactions must outweigh the entropic cost of fixing the chain into a unique structure. Strategies used for the designing of protein structure: Many different strategies have been used to achieve this goal. Most of these strategies have expanded considerable effort to maximize the strength and number of favorable interactions in the designed structure. The entropic cost of folding is reduced by introducing covalent cross-links, which limit the number of conformational states accessible to the chain. Following are the different strategies that have been employed to design novel protein structure:A. Self-Assembly of Modular Units of Secondary Structure Is the simplest and most direct strategy In modular approach a single unit of secondary structure i.e. α-structure and β-structure is synthesized as an individual segment of poly peptide chain. β-Structures are somewhat less modular than α-structures because individual β-strands are not stable in isolation they must be linked together by interstrand hydrogen bonds. Self assembly is mediated by non-covalent interactions that are designed into the peptide. The key advantage of this approach is its simplicity, both at the level of design and at the level of synthesis. The key disadvantage of this approach is the stability of structure and repetitive structures. B. Ligand-Induced Assembly Is the second strategy that uses to design novel protein employ a ligand Ligand is the metal ion that induces the assembly of modular protein segments. A ligand binding site is designed into the proposed structure at the interface of several interacting segments. If the site have a high affinity for the ligand, then the favorable free energy associated with binding the ligand will be sufficient to overcome the entropic cost and drive the peptide to self-assembled. If the peptide was synthetic in origin, the binding site can be constructed from moieties other than those represented by the 20 naturally occurring amino acids side chains. Metal ion-assisted spontaneous self-assembly of a polypeptide into a triplehelix bundle protein. Metal-Directed Protein Self-Assembly, is utilizes the simultaneous stability, lability and directionality of metal-ligand bonds to drive protein-protein interactions. The use of metal coordination to control protein self-assembly is attractive from both structural and functional perspectives: whereas the directionality and symmetry inherent in metal coordination can govern overall supramolecular geometry, the resulting interfacial metal centers may potentially offer new reactivities within biological scaffolds. Synthetic metal coordinating functionalities have previously been employed for stabilizing coiled-coil assemblies constructing reactive metal binding sites in protein interiors and tuning the potentials of redox centers, among others. It was found that in the absence of added metal (i) the helical structure are only marginally stable and (ii) this stability was concentration dependent C. Assembly of peptides via Covalent cross-linking As we know that the major obstacle in the designing of a novel protein is the conformational entropy of the polypeptide chain. When structure are assembled from several unlinked chains this entropic barrier is all the more difficult to overcome. Therefore reorganizing the peptides by covalently linking them together is a powerful strategy to direct the formation of a desired structure. The disulfide bond is the only bond that is used by the nature to cross-linked for peptides. If a designed protein is made synthetically, then a variety of other cross-links are also possible. Example: A novel cross-linker called DAB in their initial design of Betabellin, an eight-stranded β-barrel intended to fold with a simple up-and-down topology. The covalent bond (also termed a peptide bond) has unique properties that make the protein eminently suited for its role in their structure. The peptide bond has limited rotational freedom because of the partial double-bond character of the amide moiety, while the remaining bonds in the polymer main chain can rotate freely . Therefore, one in every three bonds in the chain is fixed. Because of this constraint, the polypeptide backbone is significantly more structured than typical synthetic polymers, such as polyethylenes and polyesters, which lack such rigidifying elements. Furthermore, the amide bond is equipped with both hydrogen-bond donors and acceptors, allowing the peptide backbone to engage in intricate networks of noncovalent hydrogen-bonding interactions. While these forces are inherently far weaker than the covalent bonds of the polymer chain, the additive effect of a large number of such interactions makes the folded, three-dimensional structures of proteins relatively stable. In fact, some proteins have evolved an unusual stability that allows them to retain their structure under the extreme heat and pressure conditions of deep-sea geothermal vents Fig: Protein secondary structure. The hydrogen bonding network is shown in the ball-and-stick diagram, and the backbone chain in the ribbon diagram. (a) Structure of α-helix. (b) Structure of β-hairpin The side chain amino group of lysine or ornithine and the side chain carboxylic acid groups of Glu or Asp can form peptide bonds with each other or with the N-and C- termini of the main chain. Such cross-linking leads to branched structures that are topologicaly quite different from ribosomally produced natural proteins. An advantage of these structure is that the interchain cross-links constrain the position of the chains relative to one another, and thereby reduce the entropic cost of forming a unique structure. Figure: Ribbon diagrams showing (a) the three-helix bundle fold in a designed, 73-residue peptide (the peptide structure determination was performed using highresolution NMR in aqueous solutions (PDB code, 2A3D)) and (b) a theoretical structural model for Betadoublet, a 33-residue peptide designed to dimerize into a â-sandwich structure tethered by an intermolecular disulfide bond (in black) (PDB code, 1BTD). D. Assembly of Peptides on a Synthetic Template The de novo design of polypeptide sequences with a three-dimensional structure necessary for many biological functions is limited by the complex folding process, or ‘protein folding problem’. This problem can be bypassed through constructing protein-like molecules with a ‘built-in’ device for intramolecular folding, that is, proteins of non-natural chain architecture (template-assembled synthetic proteins, TASP). Topological templates have become a versatile tool for inducing and stabilizing secondary structures (protein loops, β-turns, α-helices, β-sheets) and are widely adopted design elements for the construction of protein-like molecules, exhibiting interesting structural and functional properties. An alternate approach to the template-mediated association of peptide chains is the use of disulfide bridges to tether peptide modules and induce local folding. This strategy was utilized in the classical design of some members of the betabellin family: betadoublet176 and betabellins 14D128 and 15D. A less coercive approach to assembled proteins involves metal-mediated association of synthetic modules, whereby strong metal-peptide interactions would drive assembly. Such an approach necessitates the presence of a metal ligand in each interacting subunit and further dictates that such complexes be exchange-inert. Both synthetic peptide assemblies exhibit cooperativity in their denaturation by guanidium hydrochloride, which is a feature associated with compactly folded structures. E. Linear poly peptides that fold into Globular Structure Is the ultimate goal in the designing of novel structure of proteins three dimensional structures without the assistance of templates or covalent crosslinks. In which a polypeptide folds into its characteristic and functional threedimensional structure from random coil. Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino acids. This polypeptide lacks any developed threedimensional structure (the left hand side of the neighboring figure). Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein (the right hand side of the figure), known as the native state. The first example of a single-chain polypeptide successfully designed to fold into a stable globular structure was the α4 structure. Protein design involves a delicate balance between stabilizing a designed structure and destabilizing competing structure. The process of designing against alternative structure is sometimes called “negative design” F. Protein Design by Binary Pattering of Polar and Non-polar amino Acids Numerous studies of natural proteins have demonstrated that protein structures are remarkably tolerant to amino acid substitutions. Thus, many different amino acids can encode the information necessary to produce a given three dimensional structure. We have taken advantage of this tolerance to develop a general strategy for protein design. The strategy—called “the binary code” strategy—is based on the premise that the appropriate patterning of polar and nonpolar residues can direct a polypeptide chain to fold into elements of secondary structure, while simultaneously allowing the burial of nonpolar amino acids in a desired tertiary structure.