The genetic code--more than just a table.

The standard codon table is a primary tool for basic understanding of molecular biology. In the minds of many, the table's orderly arrangement of bases and amino acids is synonymous with the true genetic code, i.e., the biological coding principle itself. However, developments in the field reveal a much more complex and interesting picture. In this article, we review the traditional codon table and its limitations in light of the true complexity of the genetic code. We suggest the codon table be brought up to date and, as a step, we present a novel superposition of the BLOSUM62 matrix and an allowed point mutation matrix. This superposition depicts an important aspect of the true genetic code-its ability to tolerate mutations and mistranslations.

Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated.

Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.

The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion.

Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino-terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low-pH conformation of HA2, FHA2 still induces lipid mixing between liposomes in a low-pH-dependent manner. Here, we found that FHA2 induces lipid mixing between bound cells, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature, 4 degrees C. The same genetic modification of the fusion peptide inhibits both HA- and FHA2-fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low-pH conformation of the whole HA prior to fusion. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH.

15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions.

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.

Grabbing the cat by the tail: manipulating molecules one by one.

Methods for manipulating single molecules are yielding new information about both the forces that hold biomolecules together and the mechanics of molecular motors. We describe here the physical principles behind these methods, and discuss their capabilities and current limitations.

A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5' end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5' end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 A, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5' displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion.

To better understand the roles of different regions of influenza hemagglutinin in membrane fusion, we have studied the fusion properties of large unilamellar vesicles in the presence of constructs comprising the 127 amino acid ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 containing single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-terminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equimolar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 present, even at very low molar concentrations, whereas much lower rates were observed using the shorter constructs: SHA2, the fusion peptide, and the loop peptide. Concentrations of FHA2 which promoted extensive lipid mixing also induced leakage of aqueous contents. Marked effects of FHA2 were also observed with liposomes of egg phosphatidylcholine. All of the changes observed with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in promoting lipid mixing and demonstrate the contribution of other regions of the ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH dependence of fusion is not due solely to changes in the interactions between the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not required to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residues in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipid mixing activity of mutant forms of FHA2.

The synaptic SNARE complex is a parallel four-stranded helical bundle.

The heterotrimeric synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of the synaptic vesicle-associated membrane protein 2 (VAMP2) and presynaptic plasma membrane proteins SNAP-25 (synaptosome-associated protein of 25,000 Mr) and syntaxin 1A, is a critical component of the exocytotic machinery. We have used spin labeling electron paramagnetic resonance spectroscopy to investigate the structural organization of this complex, particularly the two predicted helical domains contributed by SNAP-25. Our results indicate that the N- and C-terminal domains of SNAP-25 are parallel to each other and to the C-terminal domain of syntaxin 1A. Based on these findings, we propose a parallel four-stranded coiled coil model for the structure of the synaptic SNARE complex.

The mechanism for low-pH-induced clustering of phospholipid vesicles carrying the HA2 ectodomain of influenza hemagglutinin.

Homotrimeric hemagglutinin (HA) is one of the major spike membrane glycoproteins of the influenza virus. Initial pH-triggered conformational changes in the target membrane-interacting HA2 domain are necessary for a preliminary step in membrane fusion. Using spin-labeling electron paramagnetic resonance (EPR) spectroscopy, we examined subsequent pH-dependent changes of a membrane-bound HA2 construct (FHA2, aa 1-127). Residues 91-94, 108-115, 122, and 125 were mutated to cysteine and spin-labeled. Low solvent accessibility and side chain mobility were observed by EPR at positions 91-94, 122, and 125. Spin-labels at residues 108-115 were solvent-exposed and highly mobile, revealing the presence of a flexible loop. These results are consistent with the low-pH crystal structure of a truncated HA2 domain, particularly the unusual kink loop at residues 108-115 [Bullough et al. (1994) Nature (London) 371, 37-43]. Most interestingly, at endosomal pH, spin-labels at 108-115 become immobile and no longer solvent-exposed, and this change is reversible upon reneutralization. However, little change in the EPR line shape and accessibility of spin-labels was observed in other regions. This observation implies that the FHA2 trimers interact reversibly via this specific loop, most likely in an intermolecular fashion. Furthermore, this interaction correlates well with a reversible pH-dependent clustering of FHA2-bearing vesicles evidenced by the reversible increase in turbidity and further confirmed in detail by electron microscopy. The implications of this reversible, pH-dependent interaction between FHA2 trimers are discussed in light of recent fusion models.

The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR.

Hemagglutinin (HA) is a homotrimeric surface glycoprotein of the influenza virus. In infection, it induces membrane fusion between viral and endosomal membranes at low pH. Each monomer consists of the receptor-binding HA1 domain and the membrane-interacting HA2 domain. It has been known that the NH2-terminal region of the HA2 domain, the so-called "fusion peptide", inserts into the target membrane and plays a crucial role in triggering fusion between the viral and endosomal membranes. A major portion of the HA2 domain (FHA2: aa 1 to 127) of influenza virus X-31, including the NH2-terminal fusion peptide region, was expressed in Escherichia coli. Through site-directed mutagenesis, eight cysteine (Cys) mutants in the fusion peptide region of HA2 (A5C, I6C, A7C, G8C, I10C, N12C, G13C, W14C) were generated and modified with a nitroxide spin label. Using spin-labeling electron paramagnetic resonance (EPR) techniques, we investigated the conformation, membrane topology and the local oligomeric state of the fusion peptide region in the membrane. EPR spectra showed that this region is likely to exist as a flexible monomer in the membrane at both neutral and fusogenic pH conditions. In addition, EPR power saturation methods allowed us to measure the depth in the membrane of the spin label at each mutation site. The resulting depth profile is consistent with an alpha-helix tilted approximately 25 degrees from the horizontal plane of the membrane with a maximum depth of 15 A from the phosphate group. The tilt and rotational orientation correlates well with a calculated amphiphilicity of this region.

On the dynamics and conformation of the HA2 domain of the influenza virus hemagglutinin.

To investigate the dynamics and conformation of the membrane-interacting HA2 domain of the hemagglutinin protein of influenza virus, the peripheral part of the HA2 domain (aa 1-127) was expressed in Escherichia coli. Four consecutive single-cysteine mutants, F63C, H64C, Q65C, and I66C, were generated using site-directed mutagenesis. This region is proposed to undergo a conformational change from a loop to a helical coiled-coil when going from the native to the fusion-active state [Bullough et al. (1994) Nature (London) 371, 37-43]. In the trimeric coiled-coil geometry positions 63 and 66 belong to the core so that cysteines from individual monomers are spatially close. On the other hand, positions 64 and 65 face the aqueous phase so that cyteines from monomers are spatially remote. The mutants were studied with cysteine-cysteine cross-linking and the spin-labeling electron paramagnetic resonance (EPR) in both the membrane-bound state and in the detergent-solubilized state. Extensive intramolecular cysteine-cysteine cross-linking was observed not only for F63C and I66C but also for H64C. Rates of cross-linking were comparable for these three mutants at physiological temperatures. These results are inconsistent with what is expected for a well-defined coiled-coil and suggest that the region containing the mutation sites is flexible. However, a characteristic cross-linking pattern consistent with a well-defined coiled-coil developed at very low temperatures. Line shapes of EPR spectra also indicate that this region is dynamic at ambient temperatures. Such flexibility perhaps arises from an equilibrium between a coiled-coil and a random coil conformation. No significant changes of the EPR spectra were observed upon lowering the pH to fusogenic conditions, suggesting that this flexible structure is the stable conformation at both neutral and low pH. The dynamic flexibility of this region may have important implications for the mechanism of HA-induced membrane fusion; for example it may be required for the apposition of the viral and endosomal membranes.