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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.