Proteins combinatorial

The space to be searched in protein combinatorial chemistry experiments is extremely large. Consider, for example, that a relatively short lOO-amino acid protein domain were to be evolved. The number of possible amino acid sequences of this length is 20 ° 10 ", since there are 20 naturally occurr-... 

The sequence space of proteins is extremely dense. The number of possible protein sequences is 20. It is clear that even by the fastest combinatorial procedure only a very small fraction of such sequences could have been synthesized. Of course, not all of these sequences will encode protein stmctures which for functional purjDoses are constrained to have certain characteristics. A natural question that arises is how do viable protein stmctures emerge from the vast sea of sequence space The two physical features of folded stmctures are (l)in general native proteins are compact but not maximally so. (2) The dense interior of proteins is largely made up of hydrophobic residues and the hydrophilic residues are better accommodated on the surface. These characteristics give the folded stmctures a lower free energy in comparison to all other confonnations. 

Fig. 8. A simplified combinatorial approach to identify the binding sequence for a (O) protein of interest, within 4096 sequences where ( ) represents the PCR primers, N a nucleotide, ie. A, G, C, or T. An essential aspect of this experiment is the abiHty to separate protein-bound from free DNA prior to...

Eucaryotes have many more genes and a broader range of specific transcription factors than procaryotes and gene expression is regulated by using sets of these factors in a combinatorial way. Eucaryotes have found several different solutions to the problem of producing a three-dimensional scaffold that allows a protein to interact specifically with DNA. In the next chapter we shall discuss some of the solutions that have no counterpart in procaryotes. However, the procaryotic helix-turn-helix solution to this problem (see Chapter 8) is also exploited in eucaryotes, in homeodomain proteins and some other families of transcription factors. 

The ability of the leucine zipper proteins to form heterodimers greatly expands the repertoire of DNA-binding specificities that these proteins can display. As illustrated in Figure 10.19, for example, three distinct DNA-binding specificities could, in principle, be generated from two types of monomer, while six could be created from three types of monomer and so on. This is an example of combinatorial control, in which combinations of proteins, rather than individual proteins, control a cellular process. It is one of the most important mechanisms used by eucaryotic cells to control gene expression. 

Combinatorial methods are often referred to as in vitro or directed evolution techniques. In nature, the random DNA mutations that lead to changes in protein sequences occur rarely and so evolution is usually a slow... 

EMPl, selected by phage display from random peptide libraries, demonstrates that a dimer of a 20-residue peptide can mimic the function of a monomeric 166-residue protein. In contrast to the minimized Z domain, this selected peptide shares neither the sequence nor the structure of the natural hormone. Thus, there can be a number of ways to solve a molecular recognition problem, and combinatorial methods such as phage display allow us to sort through a multitude of structural scaffolds to discover novel solutions. 

In addition to being a remarkable demonstration of the power of computer-based combinatorial design of a protein fold, this designed peptide is the shortest known peptide consisting entirely of naturally occurring amino acids that folds into a well-ordered structure without metal binding, oligomerization or disulfide bond formation. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Precisely defined collections of different chemical compounds are denominated as chemical libraries that can be efficiently prepared by methods of combinatorial chemistry. Each chemical compound owes specific structural, steiic, and electronic properties that determine all possible interactions of the small molecule with a given protein or receptor. The molecule s properties are based on the steiic arrangement of functional groups, including the conformations that can be attained by a specific structure. 

Complex optimization of the ligand-protein interactions require to scan large areas of the chemical space. Thus, the combinatorial chemist aims not at the preparation of single compounds but of chemical libraries. Chemical libraries can be produced as collections of single compounds or as defined mixtures. 

Since drug development has turned into a systematic and rational task of optimizing molecules and their interactions with proteins, cells, and organisms, combinatorial chemistry has become a significant part of this endeavor. Combinatorial methods are mainly employed in the initial (preclinical) stages of drug development. 

A constellation of genes code for PBPs of varying amino acid sequences and functionalities. PBPs occur as free-standing polypeptides and as protein fusions. This combinatorial system of structural modules results in a large increase in diversity. 

The protein structure prediction problem refers to the combinatorial problem to calculate the 3D structure of a protein from its sequence alone. It is one of the biggest challenges in structural bioinformatics. 

Finally, the region of accessible protein sequence space was extended by developing a modified version of Stemmer s combinatorial multiple-cassette mutagenesis (CMCM)... 

The field of synthetic enzyme models encompasses attempts to prepare enzymelike functional macromolecules by chemical synthesis [30]. One particularly relevant approach to such enzyme mimics concerns dendrimers, which are treelike synthetic macromolecules with a globular shape similar to a folded protein, and useful in a range of applications including catalysis [31]. Peptide dendrimers, which, like proteins, are composed of amino acids, are particularly well suited as mimics for proteins and enzymes [32]. These dendrimers can be prepared using combinatorial chemistry methods on solid support [33], similar to those used in the context of catalyst and ligand discovery programs in chemistry [34]. Peptide dendrimers used multivalency effects at the dendrimer surface to trigger cooperativity between amino acids, as has been observed in various esterase enzyme models [35]. 

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