| 1. |
- Mishima, Eriko, et al.
(författare)
-
The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel
- 2016
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 473, s. 4361-4372
-
Tidskriftsartikel (refereegranskat)abstract
- Voltage-dependent K+ (K-V) channels control K+ permeability in response to shifts in the membrane potential. Voltage sensing in K-V channels is mediated by the positively charged transmembrane domain S4. The best-characterized K-V channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K+ channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp(72) in S2 and Glu(93) in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K+ channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.
|
|
| 2. |
- Zhang, Wei, et al.
(författare)
-
Multidimensional epistasis and fitness landscapes in enzyme evolution
- 2012
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 445, s. 39-46
-
Tidskriftsartikel (refereegranskat)abstract
- The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the imitations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.
|
|
