This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12). In addition to identifying residues important for lactose transport function, the analysis revealed that numerous mutations made lac permease highly toxic to cells. In the most extreme cases, production of such proteins at very low steady-state levels reduced cell viability greater than 10 4 -fold. Both frameshift and missense mutations led to toxicity, with the frameshift mutations having the strongest effects observed. The toxic missense mutations corresponded to changes in TM12 expected to interfere with membrane insertion or folding, such as the introduction of charged residues or prolines in the putative helix. The results suggest that cellular toxicity may be a relatively common consequence of mutations altering integral membrane protein folding. An analogous toxicity might contribute to the pathogenesis of several degenerative diseases caused by mutant membrane proteins, such as retinitis pigmentosa, Charcot-Marie-Tooth syndrome, and Alzheimer's disease.Several diseases caused by mutant integral membrane proteins show dominant inheritance and are associated with tissue degeneration. These diseases include autosomal dominant retinitis pigmentosa, Charcot-Marie-Tooth disease, and Dejerine-Sottas syndrome (1-3). A simple hypothesis to help account for the pathogenesis of such diseases is that they reflect a general phenomenon in which mutations render membrane proteins directly toxic to cells (4).This report describes a mutational analysis of the 12th transmembrane segment (TM12) 1 of Escherichia coli lac permease (5, 6). Our studies identify a set of TM12 residues that tolerate a variety of substitutions without loss of transport activity. These residues show an ␣-helical periodicity and may correspond to a side of the TM12 helix which faces the lipid bilayer. A number of missense and frameshift mutations were also identified which render lac permease toxic to cells. The toxic missense changes did not cluster on either the tolerant or sensitive face of TM12 and corresponded to changes expected to cause misfolding of the mutant proteins. MATERIALS AND METHODSBacteria and Plasmids-The strains and plasmids used in this study are listed in Table I. Plasmid pCS112 is a pBR322-based plasmid derived from pCM472 (7) which carries a modified lacY with a UGA nonsense codon corresponding to codon 386 and a StyI restriction site at the NH 2 -terminal end of the sequence encoding TM12 (Table I). To construct pCS112, a SspI-SspI fragment that included phoA was first deleted from pCM472. Second, the StyI site in the tet gene was removed by StyI cleavage, followed by DNA polymerase I Klenow fragment and T4 DNA ligase treatments. Third, a StyI cleavage site was introduced at a position corresponding to the NH 2 -terminal end of TM12, and a UGA termination codon (codon 386) and SpeI cleavage site were introduced within the sequence corresponding to TM12 by site-directed muta...

The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R 30 , the ratio between a protein's DSF intensity at 30 °C and at T m . This hybrid model is shown to be more powerful than sequence-based prediction alone -and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.

Two families with hereditary sex-linked nystagmus are reported. In 1 family there were 9 males affected in 6 generations and in the other 5 males in 5 generations. The hereditary characteristic is presumably carried on the X chromosome as in red-green color blindness and hemophilia. The characteristics of the syndrome are discussed.