Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface

It is rare for amino-acid substitutions on the surface of proteins to have large stabilizing or destabilizing effects. Nevertheless, one substitution of this type, the Tyr 26----Cys mutation in lambda Cro, increases the melting temperature of the protein by 11 degrees C and the stability by 2.2 kcal mol-1. Here we show that the stability of Cro can be increased by many different amino-acid substitutions at position 26, with increasing stability showing a good correlation with decreasing side-chain hydrophobicity. As Tyr 26 is hyper-exposed to solvent in the Cro crystal structure, we suggest that wild-type and variant proteins with other hydrophobic side chains at position 26 are destabilized as a result of a reverse hydrophobic effect caused by the side chain being more exposed to solvent in the native than in the denatured state.     

Contribution of hydrophobic interactions to protein stability

A major factor in the folding of proteins is the burying of hydrophobic side chains. A specific example is the packing of alpha-helices on beta-sheets by interdigitation of nonpolar side chains. The contributions of these interactions to the energetics of protein stability may be measured by simple protein engineering experiments. We have used site-directed mutagenesis to truncate hydrophobic side chains at an alpha-helix/beta-sheet interface in the small ribonuclease from Bacillus amyloliquefaciens (barnase). The decreases in stability of the mutant proteins were measured by their susceptibility to urea denaturation. Creation of a cavity the size of a -CH2-group destabilizes the enzyme by 1.1 kcal mol-1, and a cavity the size of three such groups by 4.0 kcal mol-1.     

A new way of enhancing the thermostability of proteases

Thermostability of enzyme can be enhanced by single amino acid substitutions. Recent advances in genetic engineering have made it possible to create novel proteins in a predictable manner where structural information for the protein is available. This 'protein engineering' has already been used to enhance enzyme thermostability, but it is usually not clear which amino acid substitutions should be made. We consider that the following approach should be helpful in engineering proteins with enhanced thermostability: highly conserved residues should be left unchanged; the sequences of known mesophilic and thermophilic proteins should be used to suggest the kinds of changes likely to increase thermostability; and substitutions should be made that increase internal hydrophobicity and that stabilize helices for strong internal packing. We describe here the use of this approach to alter the thermostability of the thermostable neutral protease of Bacillus stearothermophilus, the sequence of which is known. Surprisingly we find that a single mutation that decreases thermostability can require two mutations that increase stability to compensate for it. The effects on stability are not additive, suggesting cooperativity.     

Primary structure of the alpha-subunit of transducin and its relationship to ras proteins

A group of membrane-associated guanine nucleotide binding proteins (G-proteins) are essential for transducing signals generated at cell-surface receptors into changes in cellular function and metabolism. These proteins are a complex of three subunits designated alpha, beta and gamma. The alpha-subunit is responsible for binding guanine nucleotides and seems to be characteristic of each protein. Transducin, a member of this protein family, mediates visual transduction by coupling the signal of photolysed rhodopsin with activation of a cyclic GMP phosphodiesterase. We have now cloned and sequenced the complementary DNA encoding the alpha-subunit of bovine retinal transducin and from this we have deduced the complete amino-acid sequence. The transducin alpha-subunit shares several homologous amino-acid sequences with ras gene products. The homologous segments correspond mostly to the regions thought to be involved in the guanine nucleotide binding and GTPase activity of ras proteins and to the ADP-ribosylation sites of the transducin alpha-subunit.     

Hydrogen bonding and biological specificity analysed by protein engineering

The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5-1.5 kcal mol-1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further approximately 3 kcal mol-1.     

Effect of alanine versus glycine in alpha-helices on protein stability.

The rational design of proteins requires knowledge of the helix-forming propensities (s-values) of the different amino acids. There is, however, considerable controversy about the relative values for alanine and glycine. We find from experiments on mutants of barnase that the relative effect of Ala versus Gly on helix stability depends crucially on the position in the helix (whether they are at the ends (caps) or are internal) and the context (the influence of their neighbours). Glycine is greatly preferred at the N and C caps. At internal positions, Ala stabilizes the helix relative to Gly by 0.4 to 2 kcal mol-1. The variation results from a combination of burial of hydrophobic surface on folding and interference with hydrogen bonding of the protein with solvent. There is a good empirical correlation between the relative stabilizing effects of Ala and Gly with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Gly to Ala. It is not valid to assign to each amino acid a unique s-value that is generally applicable to all positions in all helices in all proteins.     

Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain

The alpha-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pKas of ionizing groups. One of the two histidine residues (His18) in barnase, the small ribonuclease from Bacillus amyloliquefaciens, is located at the negatively charged end (C-terminal) of an alpha-helix. From NMR titrations of wild-type and engineered mutants we find that the pKa of His18 is 7.9 in wild-type enzyme, 1.6 units above the value in the urea-denatured enzyme and in model peptides. This implies that there is a favourable interaction between the protonated form of His18 and the alpha-helix that should stabilize the native structure at neutral pH by 2.1 kcal mol-1. Denaturation at various values of pH of wild-type and muant enzymes engineered at position 18 shows that this is so. The increase in stability of the enzyme as the pH changes from 8.5 to 6.3 is attributable to this interaction, and the pH-stability curve fits pKa values for His18 in native and urea-denatured enzymes that are consistent with the NMR data.     

Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3

Replacing the isoleucine at amino-acid position three of bacteriophage T4 lysozyme causes changes in the thermodynamic stability of the protein that are directly related to the hydrophobicity of the substituted residue. Structural analysis confirms that the hydrophobic stabilization is proportional to the reduction of the surface area accessible to solvent on folding.     

A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. We have isolated extragenic suppressors of the prp8-1 mutation of S. cerevisiae and present here the preliminary characterization of one of these suppressors, spp81. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed.     

A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast

Heat-shock proteins have been implicated in assembly of protein complexes, correct protein folding and uptake of proteins into organelles. In Escherichia coli, the heat-shock protein DnaJ and the Hsp70 homologue, DnaK, act together to disassemble a protein complex involved in bacteriophage lambda replication. We report the identification of SCJ1, a gene in the yeast Saccharomyces cerevisiae that encodes a homologue of the bacterial DnaJ protein. SCJ1 was identified by a genetic screen in which increased expression of candidate genes results in missorting of a nuclear-targeted test protein. The predicted amino-acid sequence of SCJ1 is 37% identical to the entire E. coli DnaJ protein. Hybridization experiments indicate that there is a family of yeast genes related to SCJ1. These findings suggest that the Hsp70 DnaK-DnaJ interaction is general to eukaryotes.     

RNA helicase activity associated with the human p68 protein

It has been proposed that p68, a nuclear protein of relative molecular mass 68,000, functions in the regulation of cell growth and division. A complementary DNA analysis of the protein has revealed extensive amino-acid sequence homology to the products of a set of genes recently identified in organisms as diverse as Escherichia coli and man, which include the eukaryotic translation initiation factor elF-4A. The protein products of the new gene family have several motifs in common which are thought to be involved in nucleic acid unwinding. As yet, however, only elF-4A, through its effect on RNA, has been shown to possess unwinding activity. Here we report that purified p68 also exhibits RNA-dependent ATPase activity and functions as an RNA helicase in vitro. The protein was first identified by its specific immunological cross reaction with the simian virus 40 large T antigen, the transforming protein of a small DNA tumour virus. Surprisingly, T antigen also has an RNA-unwinding activity: the homology between the two polypeptides, although confined to only a small region resembling the epitope of the cross-reacting antibody (PAb204), should therefore be of functional significance. Furthermore, the RNA-unwinding activity may be involved in the growth-regulating functions of both proteins.     

Comparison of alpha-tropomyosin sequences from smooth and striated muscle

Tropomyosins are a closely related family of proteins with a dimeric alpha-coiled-coil structure. Skeletal isoforms are composed of two types of subunits, alpha and beta which, in turn, are assorted into two main molecular species alpha alpha and alpha beta. Both isoforms are present in different molar ratios in individual skeletal muscle types. In small mammals, however, only alpha-chain is expressed in cardiac muscle. Tropomyosin, in association with the troponin complex (troponin-I, -T and -C) plays a central role in the Ca2+-dependent regulation of vertebrate striated muscle contraction. On the other hand, despite structural similarities with the striated isoforms, the function of this protein in smooth muscle and non-muscle cells remains unknown, because in these cells contraction is thought to be regulated by myosin-linked processes independently of tropomyosin. Here we report the nucleotide sequences of cloned complementary DNAs for rat striated and smooth muscle alpha-tropomyosin. Comparison of the derived amino-acid sequences reveals the existence of tissue-specific peptides that delimit the putative troponin-I and troponin-T binding domains of tropomyosin. S1-nuclease mapping studies reveal the existence of three distinct alpha-tropomyosin messenger RNA isoforms each encoding a different protein; these isoforms are tissue-specific, developmentally regulated and most probably encoded by the same gene.     

Growth hormone receptor and serum binding protein: purification, cloning and expression

A putative growth hormone receptor from rabbit liver and the growth hormone binding protein from rabbit serum have the same amino-terminal amino-acid sequence, indicating that the binding protein corresponds to the extracellular hormone-binding domain of the liver receptor. The complete amino-acid sequences derived from complementary DNA clones encoding the putative human and rabbit growth hormone receptors are not similar to other known proteins, demonstrating a new class of transmembrane receptors.     

Four ATP-binding sites in the midregion of the beta heavy chain of dynein.

The 'motor' proteins of eukaryotic cells contain specialized domains that hydrolyse ATP to produce force and movement along a cytoskeletal polymer (actin in the case of the myosin family; microtubules in the case of the kinesin family and dyneins). There are motor-protein superfamilies in which each member has a conserved force-generating domain joined to a different 'tail' which conveys specific attachment properties. The minus-end-directed microtubule motors, the dyneins, may also constitute a superfamily of force-generating proteins with distinct attachment domains. Axonemal outer-arm dynein from sea urchin spermatozoa is a multimeric protein consisting of two heavy chains (alpha and beta) with ATPase activity, three intermediate chains and several light chains. Here I report the sequence of cloned complementary DNA encoding the beta heavy chain of a dynein motor molecule. The predicted amino-acid sequence reveals four ATP-binding consensus sequences in the central domain. The dynein beta heavy chain is thought to associate transiently with a microtubule during ATP hydrolysis, but the ATP-dependent microtubule-binding sequence common to the kinesin superfamily is not found in the dynein beta heavy chain. These unique features distinguish the dynein beta heavy chain from other motor protein superfamilies and may be characteristic of the dynein superfamily.     

Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing

The controversy surrounding the idea that neutral mutations dominate protein evolution is attributable in part to the inadequacy of the tools available to evolutionary investigators. With a few exceptions, most investigations into the force driving protein evolution have relied on indirect criteria for distinguishing neutral and non-neutral variants. To investigate a particular pathway of molecular evolution, we have reconstructed by site-directed mutagenesis likely ancestral variants of the lysozymes of modern game birds (order Galliformes), tested their activity and thermostability and determined their three-dimensional structure. We focused on amino acids at three positions that are occupied in all known game birds either by the triplet Thr 40, Ile 55, Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins representing intermediates along the possible three-step evolutionary pathways between these triplets. Although all of these are active and stable, none of these intermediates is found in known lysozymes. A comparison of the structures and thermostabilities of the variants reveals a linear correlation between the side-chain volume of the triplet and the thermostability of the protein. Each pathway connecting the two extant triplet sequences includes a variant with a thermostability outside the range of the extant proteins. This observation is consistent with a non-neutral evolutionary pathway. The existence of variants that are more stable than the extant proteins suggests that selection for maximum thermostability may not have been an important factor in the evolution of this enzyme.     

A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.     

Isolation and characterization of the gene for a yeast mitochondrial import receptor

We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria.