Mutation, direct, alterations

Kitayama, S., Shimada, S., Xu, H., Markham, L., Donovan, D.M., and Uhl, G.R., Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding, Proc. Natl. Acad. Sci. U.S.A., 89, 7782, 1992. 

Direct immunohistochemical analysis of prostatic tissue has become very popular since the development of AR antibodies. However, a disadvantage of this technique in quantitative analysis is that the intensity of the immunohistochemical stain is dependent on the intactness of the structure of the AR. Therefore, mutations or alterations in the structure may reduce staining intensity (T5). Biochemical and immunohistochemical studies of AR content in relation to grade or stage of disease, as well as prediction of response to endocrine therapy, has been inconsistent. Nearly all primary prostate cancer specimens positively express AR protein, as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis as well as by immunohistochemical analysis on formalin-fixed, paraffin-embedded primary prostate tissues (D12, H14). In advanced-stage prostate cancer, immunohistochemical techniques has shown that metastases in bone, the... 

The use of fluorophores intrinsic to the protein allows researchers to probe protein structure and dynamics without incorporating non-native fluorophores that could perturb the native structure of the protein. Proteins usually contain one or more fluorescent amino acids, which makes dynamic studies on the native protein feasible. Often, fluorescent amino acid residues can be introduced by site-directed mutation without altering protein structure significantly. 

A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10 -fold increase in the catalytic efficiency for a nonnative substrate, valine. The crystal structure of the mutant enzyme indicated a remodeled active site and altered subunit interface caused by the cumulative effects of mutations. Most amazingly, only one of the mutations directly contacts the substrate, which underscores our limited understanding of enzyme substrate specificity. These mutations would be difficult, if not impossible, to be identified and introduced to the mutant enzyme by a rational design approach. 

The quest to determine whether P-gp contains multiple drug binding sites employed a number of distinct approaches. For example, site-directed mutagenesis revealed a multitude of residues that when mutated could alter the pattern of drug resistance conferred by P-gp [183-185]. The effects of mutations on the activity were... 

We have considered here possible effects of the protein micro-environment in modulating the properties of photosynthetic chromophores in vivo. Clearly, a combination of axial ligation, hydrogen bonding and nearby residues can define a structural scaffolding that determines the conformations of the molecules and the orientations of their substituents that, in turn, control their photophysical and photochemical characteristics. Note also that if the protein micro-environment does indeed define this scaffolding, site-directed mutations may alter the protein pocket and indirectly affect the conformations and hence the properties of the chromophores. 

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