Streptomyces avidinii протеині және оның биотинмен байланысуы

Protein in Streptomyces avidinii

Streptavidin ,/s//t//r//ɛ//p/'/t//æ//v//ɪ//d//ɪ//n/ is a 52 kDa protein (tetramer) purified from the bacterium Streptomyces avidinii. Streptavidin homo tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation constant (Kd) on the order of ≈10−14 mol/L, the binding of biotin to streptavidin is one of the strongest non covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin biotin complex's resistance to organic solvents, denaturants (e. g. guanidinium chloride), detergents (e. g. SDS, Triton X 100), proteolytic enzymes, and extremes of temperature and pH.

The crystal structure of streptavidin with biotin bound was reported by two groups in 1989. The structure was solved using multi wavelength anomalous diffraction by Hendrickson et al. at Columbia University and using multiple isomorphous replacement by Weber et al. at E. I. DuPont Central Research and Development Department. As of September 2017, there are 171 structures deposited in the Protein Data Bank. See this link for a complete list. The N and C termini of the 159 residue full length protein are processed to give a shorter ‘core’ streptavidin, usually composed of residues 13–139; removal of the N and C termini is necessary for the highest biotin binding affinity. The secondary structure of a streptavidin monomer is composed of eight antiparallel β strands, which fold to give an antiparallel β barrel tertiary structure. A biotin binding site is located at one end of each β barrel. Four identical streptavidin monomers (i. e. four identical β barrels) associate to give streptavidin's tetrameric quaternary structure. The biotin binding site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from a neighboring subunit. In this way, each subunit contributes to the binding site on the neighboring subunit, and so the tetramer can also be considered a dimer of functional dimers.

The numerous crystal structures of the streptavidin biotin complex have shed light on the origins of the remarkable affinity. Firstly, there is high shape complementarity between the binding pocket and biotin. Secondly, there is an extensive network of hydrogen bonds formed to biotin when in the binding site. There are eight hydrogen bonds directly made to residues in the binding site (the so called 'first shell' of hydrogen bonding), involving residues Asn23, Tyr43, Ser27, Ser45, Asn49, Ser88, Thr90 and Asp128. There is also a 'second shell' of hydrogen bonding involving residues that interact with the first shell residues. However, the streptavidin biotin affinity exceeds that which would be predicted from the hydrogen bonding interactions alone, suggesting another mechanism contributing to the high affinity. The biotin binding pocket is hydrophobic, and there are numerous van der Waals force mediated contacts and hydrophobic interactions made to the biotin when in the pocket, which is also thought to account for the high affinity. In particular, the pocket is lined with conserved tryptophan residues. Lastly, biotin binding is accompanied by the stabilisation of a flexible loop connecting β strands 3 and 4 (L3/4), which closes over the bound biotin, acting like a 'lid' over the binding pocket and contributing to the extremely slow biotin dissociation rate. Most attempts at mutating streptavidin result in a lowered biotin binding affinity, which is to be expected in such a highly optimized system. However, a recently engineered mutant of streptavidin, named traptavidin, was found to have more than ten fold slower biotin dissociation, in addition to higher thermal and mechanical stability. This decreased dissociation rate was accompanied by a two fold decrease in the association rate. Biotin binding affinity can be impaired by chemical labeling of streptavidin, such as with amine reactive fluorophores; flavidin is a streptavidin mutant without lysine side chains, which retains good biotin binding characteristics after such fluorescent dye labeling where the dye couples to the amino terminus.

Streptavidin is not the only protein capable of binding to biotin with high affinity. Avidin is the other most notable biotin binding protein. Originally isolated from egg yolk, avidin only has 30% sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure. Avidin has a higher affinity for biotin (Kd ~ 10−15M) but in contrast to streptavidin, avidin is glycosylated, positively charged, has pseudo catalytic activity (avidin can enhance the alkaline hydrolysis of an ester linkage between biotin and a nitrophenyl group) and has a higher tendency for aggregation. On the other hand, streptavidin is the better biotin conjugate binder; avidin has a lower binding affinity than streptavidin when biotin is conjugated to another molecule, despite avidin having the higher affinity for free, unconjugated biotin. Because streptavidin lacks any carbohydrate modification and has a near neutral pI, it has the advantage of much lower nonspecific binding than avidin. Deglycosylated avidin (NeutrAvidin) is more comparable to the size, pI, and nonspecific binding of streptavidin.