Progress in the analysis of membrane protein structure and function.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.

Large-scale purification of functional recombinant human aquaporin-2.

The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.

Gecko iota-crystallin: how cellular retinol-binding protein became an eye lens ultraviolet filter.

Eye lenses of various diurnal geckos contain up to 12% iota-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. iota-Crystallin could be either the gecko's housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with iota-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the iota-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant iota-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (approximately 1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. iota-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

The short 5' untranslated region of the betaA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning.

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. BetaA3- and betaA1-crystallin, two members of the beta-crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the betaA3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of betaA1-crystallin. It has been suggested that the very short leader (5-7 nucleotides) of the betaA3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the betaA1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both betaA3- and betaA1-crystallin at very similar relative levels.

Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions.

The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.

Truncation of betaA3/A1-crystallin during aging of the bovine lens; possible implications for lens optical quality.

During aging, extensive modifications of eye lens proteins take place, which may contribute to the development of cataract. Truncation of the accessible extensions of beta-crystallins has been suggested to be an important factor in this process. We therefore studied the truncations of bovine betaA3- and betaA1-crystallin in more detail. These proteins are identical except for the length of their N-terminal extension, 30 and 13 residues, respectively. The water-soluble and -insoluble proteins from cortex and nucleus of bovine lenses of different ages were separated by 2D-gel electrophoresis and immuno-blotted with an antiserum against betaA3. Two major truncation products were detected, which by sequence analysis were found to correspond to betaA3 having lost 11 or 22 amino acids. betaA3(-11) was only observed in the nucleus, whereas betaA3(-22) was present both in cortex and nucleus. We argue, therefore, that each of these two products is produced by a separate proteolytic enzyme. betaA3(-22) can originate by cleavage of betaA3, betaA1 and betaA3(-11). Truncation of betaA3 occurs more readily than that of betaA1, while betaA3(-11) disappears at an intermediate rate. It appears that the longer the N-terminal extension, the easier proteolysis takes place. Truncated proteins are not necessarily prone to end up in the water-insoluble fractions; other modifications leading to charge changes are more likely to be responsible for insolubilization. Truncation of the extensions of beta-crystallins could be a functional rather than a harmful process during aging of the lens; by modulating protein repulsion, it may help to maintain the protein concentration gradient that is necessary for the optical quality of the lens.

The elusive role of the N-terminal extension of beta A3- and beta A1-crystallin.

beta-Crystallins are structural lens proteins with a conserved two-domain structure and variable N- and C-terminal extensions. These extensions are assumed to be involved in quaternary interactions within the beta-crystallin oligomers or with other lens proteins. Therefore, the production of beta A3- and beta A1-crystallin from the single beta A3/A1 mRNA by dual translation initiation is of interest. These crystallins are identical, except that beta A1 has a much shorter N-terminal extension that beta A3. This rare mechanism has been conserved for over 250 million years during the evolution of the beta A3/A1 gene, suggesting that the generation of different N-terminal extensions confers a selective advantage. We therefore compared the stability and association behaviour of recombinant beta A3- and beta A1-crystallin. Both proteins are equally stable in urea- and pH-induced denaturation experiments. Gel filtration and analytical ultracentrifugation established that beta A3 and beta A1 both form homodimers. In the water-soluble proteins of bovine lens, beta A3 and beta A1 are present in the same molecular weight fractions, indicating that they oligomerize equally with other beta-crystallins. 1H-NMR spectroscopy showed that residues Met1 to Asn22 of the N-terminal extension of beta A3 have great flexibility and are solvent exposed, excluding them from protein interactions in the homodimer. These results indicate that the different N-terminal extensions of beta A3 and beta A1 do not affect their homo- or heteromeric interactions.

No significant tyrosine phosphorylation of muscle fatty acid-binding protein.

As suggested by the work on adipocyte fatty acid-binding protein (FABP), other FABPs with a tyrosine kinase consensus sequence could possibly be phosphorylated by the insulin receptor tyrosine kinase. Upon stimulation with insulin, recombinant human muscle fatty acid-binding protein (M-FABP) was phosphorylated in vitro by the insulin receptor tyrosine kinase only to a slight extent (< 0.1%). Rat soleus muscle shows at incubation autophosphorylation of insulin receptors but not phosphorylation of M-FABP after insulin stimulation. Vanadate and phenylarsine oxide had no effect on the extent of phosphorylation of M-FABP in vitro and in soleus muscle. Our results do not indicate that tyrosine phosphorylation of M-FABP is an important physiological phenomenon.