Protein bamase

Links. PDB s search engine, the Structme Explorer, can be used to retrieve PDB records, as shown in Figure 5.2. The Structure Explorer is also the primary database of links to third-party aimotation of PDB structure data. There are a number of links maintained in the Structure Explorer to Internet-based three-dimensional structure services on other Web sites. Figme 5.2 shows the Structure Summary for the protein bamase (IBNR Bycroft et al., 1991). The Structure Explorer also provides links to special project databases maintained by researchers interested in related topics, such as structural evolution (FSSP Holm and Sander, 1993), structure-structure similarity (DALI Holm and Sander, 1996), and protein motions (Gerstein et al., 1994). Links to visualization tool-ready versions of the structure are provided, as well as authored two-dimensional images that can be very helpful to see how to orient a three-dimensional structure for best viewing of certain features such as binding sites. 

The small 110 a.a. ribonuclease from B. amyloliquefaciens, bamase, has been studied extensively in A.R.Fersht s laboratory (Sali, Bycroft and Fersht, 1988 Matouschek et al, 1990 Bycroft et al., 1990 Serrano and Fersht, 1989) using NMR as an essential experimental techniqne. The df-helices of bamase have been studied with respect to stability and it was found that mutation of the THR and THR residnes located at the N-terminal of the helix (Serrano and Fersht, 1989) could destabilize the protein with up to 2.5 kcal mol b The THR and SER residnes are capable of facilitating the formation of an additional hydrogen bond in the first him of the df-helix. If THR is snbstitnted with ASP or GLU no marked change in stability was observed, probably dne to the known charge-dipole interaction between the negative charge of the ASP or GLU and the positive end of the helix macro-dipole. 

Bovine pancreatic RNase A is a member of a homologous superfamily. In addition, there is a separate family of guanine-specific microbial RNases that have evolved to have a similar active site.192,193 Ribonuclease T1 from Aspergillus oryzae and the 110-residue bamase from Bacillus amyloliquefaciens of Mr 12 392 (see Chapter 19) are the best known examples. One of the histidine residues is replaced by a glutamate in these enzymes. The microbial enzymes are much more amenable to study by protein engineering. 

Figure 19.22 The gatekeeping or filtering activity of the GroEL ATPase. The turnover number for the ATPase reaction (0.05-0.1 s-"1) is far slower than the refolding rate constant of 2-2.5 s-1 for GroEL-bound bamase. Only slowly folding proteins bind long enough to enter the chaperoning cycles of Figure 19.23.

The rate constants for the association of proteins with one another and with other macromolecules are profoundly influenced by the geometry of the interaction and by electrostatic factors. Only a small part of each protein may be involved in the formation of a protein-protein complex, which imposes a bad steric factor on the reaction. Accordingly, protein-protein association rate constants may be as low as 104 s 1 M x (Table 4.1). But there is very fast association at > 5 X 109 s-1 AT 1 at low ionic strength for proteins that have complementary charged surfaces, such as bamase with its polypeptide inhibitor barstarfwhose, properties are discussed in Chapter 19), thrombin with its polypeptide inhibitor hirudin, and ferricytochrome c with ferrocytochrome b5. 

The contribution of electrostatic interactions to fast association was analyzed by applying the classical Debye-Hiickel theory of electrostatic interactions between ions to mutants of bamase and barstar whose ionic side chains had been altered by protein engineering (Chapter 14).16 The association fits a two-step model that is probably general (equation 4.84). 

The amyloidogenic Sup35 protein has been successfully used to immobilize three enzymes bamase, carbonic anhydrase, and glutathione S-transferase (Baxa et al., 2002). Each of these enzymes was linked to the Sup35 sequence which drives assembly creating three different fusion proteins which successfully formed fibrils displaying functional enzymes on the fibril surface. 

M. Prevost, S. J. Wodak, B. Tidor, and M. Karplus, Contribution of the hydrophobic effect to protein stability Analysis based on simulations of the Ile-96 -+ Ala mutation in bamase, Proc. Natl. Acad. Sci. USA 88 10880 (1991). 

Vijayakumar M et al (1998) Electrostatic enhancement of diffusion-controlled protein-protein association comparison of theory and experiment on bamase and barstar. J Mol Biol 278(5) 1015-1024... 

Neira JL, Vazquez E, Fersht AR (2000) Stability and folding of the protein complexes of bamase. Eur J Biochem 267(10) 2859-2870... 

Buckle AM, Schreiber G, Fersht AR (1994) Protein-protein recognition crystal structural analysis of a bamase-barstar complex at 2.0-A resolution. Biochemistry 33(30) 8878-8889... 

Serrano L, Day AG, Fersht AR (1993) Step-wise mutation of bamase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. J Mol Biol 233(2) 305-312... 

Schreiber G, Fersht AR (1993) Interaction of bamase with its polypeptide inhibitor barstar studied by protein engineering. Biochemistry 32(19) 5145—5150... 

In a multi-tryptophan protein, fluorescence quantum yield of the tryptophans can be additive or not. In the first case, the others do not influence each tryptophan while in the second case mainly energy transfer to or from the other tryptophan residues can influence quantum yield. In the absence of energy transfer between the tryptophan residues, mutants with one tryptophan residue are prepared, the other tryptophan residues of the protein are replaced by phenylalanine, and quantum yield of the corresponding tryptophan in each mutant is determined experimentally by comparing its fluorescence to that of free tryptophan in solution. For example, quantum yields of tryptophan residues in Bamase have been determined in the wild type and in mutants where only one tryptophan residue has been conserved. 

Bamase is a ribonuclease synthesized by Bacillus amyloiujuefaciens and then secreted into the growth medium. The same cells synthesize barstar. a potent inhibitor of barnase. and retain it inside the cell to inhibit ai barnase that is not seethed Many studies related to the interaction between bamase and barstar have been peformed in order to understand the rules, if any, of protein-protein interactions. Bamase has three tiyptophan residues present n positions 35.71 and 94 (Figure 2.28). 

Figure 2.28. Schematic representation of the structure of bamase sliuwing the positiois of the three tiyptophan residues and His 18. Source Dc Bcuckeker. 1C. Volckant, G. and Engelborghs. Y. 1999. Proteins Structure, Functions and Genetics 36.42-S3. Aiilhonzalion of reprint accorded 1 Wiley Interscience.

Bamase, a I lO-residue extracellular protein found in bacillus amyloliquefaciens, is a nbonuclease with potentially lethal functions within tlie cell. The RNase activity of barnase is inhibited by barstar, a 90-residue polypeptide, which binds tightly to barnase through salt bridges and hydrogen bonds. 

Ababou, A., van der Vaart, A., Gogonea, V., Merz Jr., K.M. Interaction energy decomposition in protein-protein association A quantum mechanical study of bamase-barstar complex. Biophys. Chem. 2007,125,221-36. 

Bamase is an extracellular ribonuclease wUch is often used as a model ft>r protein folding. It is a idative y small protein (110 amino acids, 12.4 kDa). Bamase contans three tryptophan residues (Figure 16.44), one of which is located close to histidine-18. Examination of the three single-tryptophan mutants of bamase provided an interesting example of energy transfer between tryptophan residues and the quenching effects of the nearby histidine. "... 

Charge Complementarity in the Bamase-Barstar Protein Complex. 

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