Barnase is a bacterial protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 that consists of 110 amino acid

Amino acid

Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

s and has ribonuclease

Ribonuclease

Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.-Function:All organisms studied contain...

 activity. It is synthesized and secreted by the bacterium Bacillus amyloliquefaciens

Bacillus amyloliquefaciens

Bacillus amyloliquefaciens is a species of bacteria that is the source of the BamH1 restriction enzyme. It also synthesizes a natural antibiotic protein barnase, a widely studied ribonuclease that forms a famously tight complex with its intracellular inhibitor barstar.-Discovery and name:B...

, but is lethal to the cell when expressed without its inhibitor barstar

Barstar

Barstar is a small protein synthesized by the bacterium Bacillus amyloliquefaciens. Its function is to inhibit the ribonuclease activity of its binding partner barnase, with which it forms an extraordinarily tightly bound complex within the cell until barnase is secreted. Expression of barstar is...

. The inhibitor binds to and occludes the ribonuclease active site, preventing barnase from damaging the cell's RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

 after it has been synthesized but before it has been secreted. The barnase/barstar complex is noted for its extraordinarily tight protein-protein binding

Protein-protein interaction

Protein–protein interactions occur when two or more proteins bind together, often to carry out their biological function. Many of the most important molecular processes in the cell such as DNA replication are carried out by large molecular machines that are built from a large number of protein...

, with an on-rate of 10⁸s⁻¹M⁻¹.

Protein folding studies

Barnase has no disulfide bonds, nor does it require divalent cations or non-peptide components to fold. This simplicity, in combination with its reversible folding transition, means that barnase has been extensively studied in order to understand how proteins fold. The folding of barnase has been extensively studied in the laboratory of Alan Fersht

Alan Fersht

Sir Alan Roy Fersht FRS is a British chemist at the MRC Laboratory of Molecular Biology in Cambridge. He is distinguished for his pioneering work on protein folding.-Biography:...

, who used it as the test case in developing a method of characterizing protein folding

Protein folding

Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....

 transition state

Transition state

The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...

s known as phi value analysis

Phi value analysis

Phi value analysis is an experimental protein engineering method used to study the structure of the folding transition state in small protein domains that fold in a two-state manner. Since the folding transition state is by definition a transient and partially unstructured state, its structure is...

.

Active site and catalytic mechanism

Barnase catalyzes hydrolysis

Hydrolysis

Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 at diribonucleotide GpN sites. Cleavage occurs in two steps using a general acid-base mechanism: a cyclic intermediate is formed during the first transesterification

Transesterification

In organic chemistry, transesterification is the process of exchanging the organic group R″ of an ester with the organic group R′ of an alcohol. These reactions are often catalyzed by the addition of an acid or base catalyst...

 step, which is then hydrolysed to release the cleaved RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

. The two most important residues involved in catalysis are Glu73 and His102, which are both essential for enzymatic activity. Glu73 is the general base whilst His102 is the general acid. Although it is not directly involved in acid-base catalysis, Lys27 is also critical for activity; it has been implicated in transition-state substrate binding.