Protein design

To explore the feasibility of such an approach for the design of active catalysts, we have systematically replaced the secondary structural elements in the homodimeric helical bundle chorismate mutase (Fig. 3.18) with binary-patterned units of random sequence. Genetic selection was then used to assess the catalytic capabilities of the proteins in the resulting libraries, providing quantitative information about the robustness of this particular protein scaffold and insight into the subtle interactions needed to form a functional active site [119]. 

In the first stage of our experiment, modules that correspond to the HI and H2/H3 helices were combined individually with the appropriate complementary wild-type helical segments of the thermostable MjCM , a protease-resistant version of the dimeric M.jannaschii chorismate mutase (Fig. 3.18) [37]. This amounted to randomizing 37 % and 42 % of the entire protein for the HI and H2/H3 replacement modules, 

Catalysts from both the HI and H2/H3 libraries were isolated and characterized [119]. Despite the restricted set of building blocks used in the randomized segments, the enzymes are remarkably active, with modestly elevated Km values and kc ll values in the range 0.2 to 2.3 s which compare favorably with the value of 5.7 s 1 for wild-type MjCM [37]. While most positions in the protein are relatively tolerant to substitution, sequence analysis has identified a few sites as quite restric- 

Experiments of this kind can provide valuable insights into the chemical determinants of protein structure. The results support speculations that ancient proteins might have been constructed from small numbers of amino acids [121 - 123], Nevertheless, the relatively low abundance of active enzymes in the combinatorial libraries 

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In a different approach, a TNF fusion protein designated TNF-Selectokine has been described [5]. This TNF prodrug is comprised of a trimeric scFv-TNF fusion protein to which a TNFR fragment has been fused at the C-terminal. A flexible peptide linker between TNF and the blocking receptor domain is comprised of sequences specifically recognized by tumor associated proteases such as tissue plasminogen activator, urokinase type plasminogen activator or... 

A protein, designated cyctochrome c", isolated from the methylotrophic bacterium Methylophilus methylotrophus, has been studied extensively because of its unusual properties and was found to have an average molecular mass of 14293.0 Da and to contain 124 amino acid residues. The A-terminal sequence to residue 62 had been determined and the heme binding site had been located at Cys-49 and Cys-52 [12]. Further studies were concerned with determining the remainder of the sequence. 

Hayashi C.Y. and Lewis R.V., Spider flagelUfotm silk Lessons in protein design, gene structure, and molecular evolution, BioEssays, 23, 750, 2001. 

Other protein design methods have demonstrated comparable successes [64, 66,70]. 

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In the sarcoplasm of resting muscle, the concentration of Ca + is 10 to 10 mol/L. The resting state is achieved because Ca + is pumped into the sarcoplasmic reticulum through the action of an active transport system, called the Ca + ATPase (Figure 49-8), initiating relaxation. The sarcoplasmic reticulum is a network of fine membranous sacs. Inside the sarcoplasmic reticulum, Ca + is bound to a specific Ca -binding protein designated calsequestrin. The sarcomere is surrounded by an excitable membrane (the T tubule system) composed of transverse (T) channels closely associated with the sarcoplasmic reticulum. 

In 1985 Dr. Michael Rossmann and his colleagues determined for the first time the three-dimensional structure of a human rhinovirus [15], Their studies, performed with human rhinovirus type 14 (HRV-14), revealed the structure as an eicosahedron consisting of four proteins designated VP1, VP2, VP3, and VP4 forming a protomeric unit, combined to form a fivefold axis of symmetry (Fig. 2). The surface of the capsid... 

A human cDNA highly homologous to the transferrin receptor, has been identified and reported to encode a protein, designated TfR2, which binds diferrictransferrin and mediates iron uptake of transferrin-bound iron (Kawabata etal., 1999). A mouse orthologue of human TfR2 has been found independently (Fleming etal.,... 

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ABBOTT Bioresearch Center. ChemoCentryx. Hyseq Pharmaceuticals. Protein Design Labs. 

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