Single-residue mutations

Since the formation of amyloid fibrils involves changes in secondary structure, an obvious question is whether this occurs through partially folded intermediates or randomly structured segments. Clues to the structure of partially folded intermediates have been provided by spectroscopic studies, by the effects of single residue mutations, and by varying conditions under which intermediates form. 

Further, the removal of benzodiazepine sensitivity in a selective a subunit in a mouse using the gene knockin technique has established that the al subunit plays a major role in the sedative and amnesiac effects of benzodiazepines, part of the anticonvulsant effect and little of the anxiolytic effect the latter effects are more importantly mediated by the a2 subunit [5, 6], The 0 subunit selectivity for the drugs loreclezole (an anxiolytic) and etomidate (an anesthetic) allowed determination that a single residue in the M2 domain could account for this selectivity (02 = 03 >01). When a mouse knockin selectively removed the etomidate sensitivity of the 02 subunit, the animals showed reduced sensitivity to sedative effects of etomidate but no reduction of the true anesthetic effects. In contrast, mutation of the 03 subunit to negate etomidate sensitivity of that subunit alone resulted in a mouse with no sensitivity to the anesthesia produced by etomidate. This proved that the GABA receptor is the target of at least this one anesthetic (etomidate) and, furthermore, that the specific locations in the brain of 03 subunits are important for anesthetic action, while the... 

Interestingly, we have recently identified a mutation of a tyrosine in the third intracellular loop of the hDAT that causes a major alteration in the conformational equilibrium of the transport cycle, and thus as such is comparable to mutants on G protein-coupled receptors causing constitutive isomerization of the receptor to the active state (66). Most importantly, this conclusion is based on the observation that mutation of the tyrosine completely reverts the effect of Zn2+ at the endogenous Zn2+ binding site in the hDAT (50,51) from potent inhibition of transport to potent stimulation of transport (Fig. 6). In the absence of Zn2+, transport capacity is reduced to less than 1% of that observed for the wild-type, however, the presence of Zn2+ in only micromolar concentrations causes a close to 30-fold increase in uptake (66). Moreover, it is found that the apparent affinities for cocaine and several other inhibitors are substantially decreased, whereas the apparent affinities for substrates are markedly increased (66). Notably, the decrease in apparent cocaine affinity was around 150-fold and thus to date the most dramatic alteration in cocaine affinity reported upon mutation of a single residue in the monoamine transporters (66). 

A lethal single-base mutation in which a serine residue essential for enzyme activity is replaced by phenylalanine to give an enzymatically inactive product... 

In a different approach, Azuma et al. (1984) recombined the same heavy chain with two different light chains. The reconstituted immunoglobulins, which were identical except for a single Phe/Tyr replacement at the VXJX junction, differed in affinity for a haptenic determinant by a factor of about 100. This sequence difference is probably a result of variation in the position of VJ recombination and not of somatic mutation. Chain recombination was also used by Berek and Milstein (1987) to show that a single residue in CDR1 of a VK region can have a substantial effect on affinity. 

We recently developed a systematic method that uses the intrinsic tryptophan residue (Trp or W) as a local optical probe [49, 50]. Using site-directed mutagenesis, tryptophan can be mutated into different positions one at a time to scan protein surfaces. With femtosecond temporal and single-residue spatial resolution, the fluorescence Stokes shift of the local excited Trp can be followed in real time, and thus, the location, dynamics, and functional roles of protein-water interactions can be studied directly. With MD simulations, the solvation by water and protein (residues) is differentiated carefully to determine the hydration dynamics. Here, we focus our own work and review our recent systematic studies on hydration dynamics and protein-water fluctuations in a series of biological systems using the powerful intrinsic tryptophan as a local optical probe, and thus reveal the dynamic role of hydrating water molecules around proteins, which is a longstanding unresolved problem and a topic central to protein science. 

One of the main motivations of using synthetic DNA for cellular engineering seems to be at odds with the random nature of directed evolution. Traditionally, PCR-based methods have been used to create sequence diversity, inspired by the fact that mutations in nature commonly arise from errors in DNA replication. PCR-based methods are preferred when there is no prior knowledge about where mutations are likely to influence the traits of interest, but are limited in that the sequence diversity that results is restricted and biased. With single base mutations per codon - a common assumption with most protocols - only 5.7 amino acids are accessible per position on average, and in most cases, the resulting set of amino acids does not accurately represent the spectrum of physicochemical properties of naturally-occurring residues [76]. 

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