Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli.

Lee, Jong-In; Hwang, Pung Pung; Hansen, Camilla Hartmann Friis; Wilson, Thomas H.

doi:10.1016/s0021-9258(19)36751-1

Cited by 98 publications

(46 citation statements)

References 39 publications

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“…The remarkable ability of lac permease to tolerate paired neutral replacements in Asp237 and Lys358 or reversal of the charge pair distinguishes this interaction from that observed for Asp240 and Lys319 (Sahin-T6th et al, 1992;Lee et al, 1992). It has been shown (Sahin-T6th et al, 1992) that simultaneous replacement of Asp240 and Lys319 with Cys and/or Ala results in significant but diminished active lactose transport and that interchanging the residues leads to complete inactivation.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, the secondary structure model of the permease was altered to accommodate a putative salt bridge between Asp237 and Lys358 within the low dielectric of the membrane by shifting residues Phe247-Thr235 from the hydrophilic domain between helices VII and VIII into transmembrane helix VII (Figure 1). Subsequent studies (Sahin-T6th et al, 1992;Lee et al, 1992) also reveal a functional interaction between Asp240 and Lys319, leading to the hypothesis that putative helix VII which contains Asp237 and Asp240 neighbors helices X (Lys319) and XI (Lys358) in the tertiary structure of the permease.…”

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confidence: 99%

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Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli

Sahin–Tóth²,

Hr³

1993

Biochemistry

125

137

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Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S. C., Hansen, C. L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli

Sahin–Tóth²,

Hr³

1993

Biochemistry

125

137

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“…Since the cytoplasmic ends of helices IV and V may be in close proximity (48), it is possible that Glu126 and Arg144 interact by charge pairing. However, unlike charge pairs Asp237 (helix VII)-Lys358 (helix XI) and Asp240 (helix VII)-Lys319 (helix X), which are not required for active transport (49)(50)(51)(52)(53)(54), Glu126 and Arg144 are essential residues. Therefore, if Glu126 and Arg144 are charge paired, it cannot be demonstrated by functional assays, and it is not surprising that double neutral replacement or inversion of the charged residues between positions 126 and 144 inactivates the permease.…”

Section: Discussionmentioning

confidence: 99%

Cysteine-Scanning Mutagenesis of Helix IV and the Adjoining Loops in the Lactose Permease of Escherichia coli: Glu126 and Arg144 Are Essential

1997

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Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.

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“…Like hydrogen bonds, other electrostatic interactions have the potential to be stronger in the low dielectric membrane environment, though the same mitigating factors described above apply. Many membrane proteins require salt bridges to function properly (Lee et al , 1992; Call et al , 2002; Kim et al , 2004; Hertel et al , 2010; Cui et al , 2013), but the strength of these interactions has largely gone unstudied. One group was able to probe a set of salt bridges in the β‐barrel membrane protein OmpA (Hong et al , 2006) and found interaction free energies varying from ~−0.6 to −5 kcal/mol.…”

Section: Introductionmentioning

confidence: 99%

How physical forces drive the process of helical membrane protein folding

Corin

Bowie

2022

EMBO Reports

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Protein folding is a fundamental process of life with important implications throughout biology. Indeed, tens of thousands of mutations have been associated with diseases, and most of these mutations are believed to affect protein folding rather than function. Correct folding is also a key element of design. These factors have motivated decades of research on protein folding. Unfortunately, knowledge of membrane protein folding lags that of soluble proteins. This gap is partly caused by the greater technical challenges associated with membrane protein studies, but also because of additional complexities. While soluble proteins fold in a homogenous water environment, membrane proteins fold in a setting that ranges from bulk water to highly charged to apolar. Thus, the forces that drive folding vary in different regions of the protein, and this complexity needs to be incorporated into our understanding of the folding process. Here, we review our understanding of membrane protein folding biophysics. Despite the greater challenge, better model systems and new experimental techniques are starting to unravel the forces and pathways in membrane protein folding.

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Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli.

Cited by 98 publications

References 39 publications

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli

Cysteine-Scanning Mutagenesis of Helix IV and the Adjoining Loops in the Lactose Permease of Escherichia coli: Glu126 and Arg144 Are Essential

How physical forces drive the process of helical membrane protein folding

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