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The lipid-protein interface of a Shaker K(+) channel.

Hong KH, Miller C - J. Gen. Physiol. (2000)

Bottom Line: A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence.These results, combined with an analogous study of S2 (Monks, S., D.J.Miller. 1999.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

ABSTRACT
Tryptophan-substitution mutagenesis was applied to the first and third transmembrane segments (S1 and S3) of a Shaker-type K(+) channel for the purpose of ascertaining whether these sequences are alpha-helical. Point mutants were examined for significant functional changes, indicated by the voltage-activation curves and gating kinetics. Helical periodicity of functional alteration was observed throughout the entire S1 segment. A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence. In both helical stretches, tryptophan-tolerant positions are clustered on approximately half the alpha-helix surface, as if the sidechains are exposed to the hydrocarbon region of the lipid bilayer. These results, combined with an analogous study of S2 (Monks, S., D.J. Needleman, and C. Miller. 1999. J. Gen. Physiol. 113:415-423), locate S1, S2, and S3 on the lipid-facing periphery of K(v) channels.

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Results of Trp and Asn mutagenesis of S3. An α-helical wheel (A) and a net (B) diagram of S3 are shown as in Fig. 7. *Residues that were tested by Asn substitution. Striped circles represent ambiguous residues on the basis of Asn mutants, as discussed in text. (C) A restricted helical wheel diagram limited to the first 14 residues of S3.
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Figure 8: Results of Trp and Asn mutagenesis of S3. An α-helical wheel (A) and a net (B) diagram of S3 are shown as in Fig. 7. *Residues that were tested by Asn substitution. Striped circles represent ambiguous residues on the basis of Asn mutants, as discussed in text. (C) A restricted helical wheel diagram limited to the first 14 residues of S3.

Mentions: In stark distinction to S1, S3 substitutions fail to produce an orderly pattern of functional segregation on a helical wheel diagram (Fig. 8 A). The only trend apparent is a tendency for low-impact residues to cluster in the upper right-hand quadrant of the diagram, in rough correspondence with the location of the sequence-variable residues. The preponderance of the wheel's perimeter is festooned by high-impact residues, but several low-impact positions are scattered among these. An additional source of confusion is that three functionally tolerant positions (P322, F324, and L327) are tightly conserved among S3 segments, while five high-impact residues (M312, T326, V330, V331, and A332) show a remarkable sequence variability among polar, nonpolar, large, and small sidechains (Fig. 2 D). It is tempting to conclude from the results that S3 is largely surrounded by protein and is poorly exposed to lipid. However, this conclusion would be logically flawed, since the Trp-scanning strategy is useful only if an unforced functional periodicity emerges from the data, as with S1 and S2. The absence of a pattern is uninterpretable; in such a case, the scanned sequence might not have a periodic secondary structure at all, or it might be a helix (or any other structure) specifically packed on all sides against other parts of the protein.


The lipid-protein interface of a Shaker K(+) channel.

Hong KH, Miller C - J. Gen. Physiol. (2000)

Results of Trp and Asn mutagenesis of S3. An α-helical wheel (A) and a net (B) diagram of S3 are shown as in Fig. 7. *Residues that were tested by Asn substitution. Striped circles represent ambiguous residues on the basis of Asn mutants, as discussed in text. (C) A restricted helical wheel diagram limited to the first 14 residues of S3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887780&req=5

Figure 8: Results of Trp and Asn mutagenesis of S3. An α-helical wheel (A) and a net (B) diagram of S3 are shown as in Fig. 7. *Residues that were tested by Asn substitution. Striped circles represent ambiguous residues on the basis of Asn mutants, as discussed in text. (C) A restricted helical wheel diagram limited to the first 14 residues of S3.
Mentions: In stark distinction to S1, S3 substitutions fail to produce an orderly pattern of functional segregation on a helical wheel diagram (Fig. 8 A). The only trend apparent is a tendency for low-impact residues to cluster in the upper right-hand quadrant of the diagram, in rough correspondence with the location of the sequence-variable residues. The preponderance of the wheel's perimeter is festooned by high-impact residues, but several low-impact positions are scattered among these. An additional source of confusion is that three functionally tolerant positions (P322, F324, and L327) are tightly conserved among S3 segments, while five high-impact residues (M312, T326, V330, V331, and A332) show a remarkable sequence variability among polar, nonpolar, large, and small sidechains (Fig. 2 D). It is tempting to conclude from the results that S3 is largely surrounded by protein and is poorly exposed to lipid. However, this conclusion would be logically flawed, since the Trp-scanning strategy is useful only if an unforced functional periodicity emerges from the data, as with S1 and S2. The absence of a pattern is uninterpretable; in such a case, the scanned sequence might not have a periodic secondary structure at all, or it might be a helix (or any other structure) specifically packed on all sides against other parts of the protein.

Bottom Line: A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence.These results, combined with an analogous study of S2 (Monks, S., D.J.Miller. 1999.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

ABSTRACT
Tryptophan-substitution mutagenesis was applied to the first and third transmembrane segments (S1 and S3) of a Shaker-type K(+) channel for the purpose of ascertaining whether these sequences are alpha-helical. Point mutants were examined for significant functional changes, indicated by the voltage-activation curves and gating kinetics. Helical periodicity of functional alteration was observed throughout the entire S1 segment. A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence. In both helical stretches, tryptophan-tolerant positions are clustered on approximately half the alpha-helix surface, as if the sidechains are exposed to the hydrocarbon region of the lipid bilayer. These results, combined with an analogous study of S2 (Monks, S., D.J. Needleman, and C. Miller. 1999. J. Gen. Physiol. 113:415-423), locate S1, S2, and S3 on the lipid-facing periphery of K(v) channels.

Show MeSH
Related in: MedlinePlus