N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

The effect of a hydrophobic N-terminal probe on translational pausing of chloramphenicol acetyl transferase and rhodanese.

The effect on translational pausing of a hydrophobic probe, coumarin, at the N terminus of nascent peptides was investigated. Two different proteins, bacterial chloramphenicol acetyltransferase and bovine rhodanese, were synthesized by coupled transcription/translation in a cell-free system derived from Escherichia coli. Protein synthesis was initiated with N-formyl-Met-tRNAf or N-acetyl-S-coumarin-Met-tRNAf. Cotranslational incorporation of the coumarin derivative generated nascent polypeptides with a hydrophobic residue at their N termini. The effect of the two N-terminal groups on the size distribution and quantity of the peptides formed by translational pausing was investigated. The N-terminal coumarin caused an accumulation of nascent chloramphenicol acetyltransferase peptides in the mass range of 3.5-4.0 kDa that reflects a delay in translation at this point. No similar effect on rhodanese pause-site peptides was observed. This effect on translational pausing cannot be explained by either mRNA secondary structure or rare codons and tRNA abundance. It is suggested that the effect of N-terminal coumarin on translational pausing is the result of the interaction of the nascent peptide with components of the large ribosomal subunit along the path it follows between the peptidyl transferase center and the exit site on the distal surface.

Co-translational folding.

Nascent proteins appear to fold co-translationally. The ribosome itself may function as a chaperone, providing a sheltered environment in which the nascent peptide is protected from aggregation and degradation, and in which folding into the tertiary structure is facilitated by interactions both with ribosomal proteins and with specific segments of the ribosomal RNA.

Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression.

Rhodanese mutants containing sequential NH2-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli. Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degreesC. Immunoprecipitation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated. Cell-free transcription/translation at 37 degreesC showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo. However, at 30 degreesC in vitro, both Delta1-10 and Delta1-11 showed considerable activity. Truncations in the NH2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.

Different conformations of nascent peptides on ribosomes.

The length at which the N terminus of nascent proteins becomes available to antibodies during their synthesis on ribosomes was determined. Three different proteins, bovine rhodanese, bacterial chloramphenicol acetyltransferase and MS2 coat protein, were synthesized with coumarin at their N terminus in a cell-free system derived from Escherichia coli. A derivative of coumarin was cotranslationally incorporated as N-coumarin-methionine at the N terminus of polypeptides. The interaction of specific anti-coumarin antibodies with this N-terminal coumarin of ribosome-bound nascent peptides was examined. The results indicate that short nascent peptides of each of the three proteins are unreactive, that the length at which they become accessible to the antibodies is different for the three proteins, and that longer peptides differ in their reactivity. It is suggested that these differences are due to differences in the conformation acquired by the peptides as they are synthesized on the ribosomes.

GroEL and GroES increase the specific enzymatic activity of newly-synthesized rhodanese if present during in vitro transcription/translation.

Enzymatically active mammalian rhodanese, a mitochondrial matrix enzyme, which has been found to require assistants for efficient refolding in vitro, has been synthesized from a plasmid in a cell-free, fractionated, coupled transcription/translation system derived from Escherichia coli. The bacterial chaperonins, GroEL and GroES, along with the rhodanese substrate thiosulfate greatly enhance the specific enzymatic activity of the rhodanese polypeptide that is formed. Indirect evidence suggests that the effect of the GroEL/ES chaperonins is on ribosome-bound nascent peptides. The in vitro transcription/translation system produces sufficient amounts of rhodanese to provide a system for studying factors that control the initial steps in folding of nascent proteins.

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.

The synthesis of polyphenylalanine on ribosomes to which erythromycin is bound.

Erythromycin binds to the large subunit of Escherichia coli ribosomes at a specific site that is very close to the amino acid of aminoacyl-tRNA bound into the peptidyltransferase center, and to the site to which puromycin is bound, the P and A sites, respectively, of the classical two-site model of ribosome function. Both erythromycin and puromycin affect fluorescence from fluorescent derivatives of aminoacyl-tRNAs, while both puromycin and aminoacyl-tRNAs affect fluorescence of fluorescent derivatives of erythromycylamine. The results demonstrate unequivocally that erythromycin, deacylated tRNA, a peptidyl-tRNA analogue and puromycin can be bound simultaneously to the same ribosome. Nascent peptides of more than a few amino acids in length block binding of erythromycin to the ribosomes but, unlike most other peptides, long polyphenylalanine chains can be synthesized on ribosomes to which erythromycin is bound. It is suggested that this refractory synthesis in the presence of erythromycin reflects the atypical physical and structural properties of polyphenylalanine.

The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes.

Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin.

Thermodynamic study of domain organization in troponin C and calmodulin.

Intramolecular melting of troponin C, calmodulin and their proteolytic fragments has been studied microcalorimetrically at various concentrations of monovalent and divalent ions. It is shown by thermodynamic analysis of the experimentally determined excess heat capacity function that the four calcium-binding domains in these two related proteins are not integrated into a single co-operative system, as would be the case if they formed a common hydrophobic core in the molecule, but still interact with each other in a very specific way. There is a positive interaction between domains I and II, which is so strong that they actually form a single co-operative block. The interaction between domains III and IV is positive also, although much less pronounced, while the interaction between the pairs of domains (I and II) and (III and IV) is negative, as if they repel each other. The structure of the co-operative block of domains I and II at room temperature does not depend noticeably on the ionic conditions, which influence its stability to a small extent only. The same applies to domain IV of calmodulin, but in troponin C this domain is unstable in the absence of divalent ions, in solutions of low ionic strength. In both proteins, the least stable is domain III, which forms a compact ordered structure at room temperature only in the presence of Ca2+. In troponin C, calcium ions can be replaced by magnesium ions, although the compact structure of domain III formed by these two ions does not seem to be quite identical. Thus, at conditions close to physiological, with regard to temperature and ionic strength, the removal of free Ca2+ from the solution induces in both proteins a reversible transition of domain III to the non-compact disordered state. This dramatic Ca2+-induced change in the domain III conformation in troponin C and calmodulin might play a key role in the functioning of these proteins as a Ca2+-controlled switch in the molecular mechanisms of living systems.

[The presence of cooperative structures in tryptic fragments of troponin C]. / Nalichie kooperativnoi struktury u tripticheskikh fragmentov troponina C.

Scanning microcalorimetry has been used to study the ability of TR-1 and TR-2 tryptic fragments of troponin C to form an ordered compact structure in solution under different conditions. It has been shown that: (1) in the presence, as well as in the absence of bivalent ions both fragments have a structure which can melt with an intensive heat absorption at heating; (2) the structure of fragment TR-1 containing two Ca2+-specific domains (domains I and II) melts as a whole under all conditions studied and therefore the domains form one cooperative block. Binding of Ca2+ or Mg2+ ions stabilizes the block structure, however, significant conformational rearrangements which would lead to a change of denaturational enthalpy do not occur; (3) Ca2+Mg2+-domains of fragment TR-2 (domains III and IV) represent individual cooperative units, blocks. Stability of these cooperative blocks strongly depends on concentration of bivalent ions and in the presence of 2 mM EDTA the melting temperature of one of them is below 10 degrees. Thermodynamic melting temperature of one of them is below 10 degrees. Thermodynamic melting parameters of cooperative blocks within peptides and in the intact molecule of troponic C are compared.

[The domain organization of calmodulin]. / Issledovanie domennoi organizatsii kalmodulina.

Differential scanning microcalorimetry was used to study the domain organization of calmodulin and its fragments obtained by trypsin and thrombin treatment of the protein. It has been shown that (1) at physiological concentrations of Ca2+ ions (10(-6) divided by 10(-5) M) the protein structure represents three cooperative blocks, one of which contains two Ca2+-binding domains and the two others contain one Ca2+-binding domain; (2) stability of the cooperative blocks strongly depends on the Ca2+ concentration and in the presence of 2 mM EDTA the cooperative block containing Ca2+-binding domain III melts already at room temperature; (3) in the absence of Ca2+ ions the addition of Mg2+ or Na+ ions to the buffer system (to the concentration of 2 mM and 150 mM, respectively) does not lead to stabilization of the cooperative structure of the block containing Ca2+-binding domain III; (4) judging by thermodynamic parameters of melting, the structure of cooperative blocks within the peptides coincides with their structure in the intact molecule.

Stability of troponin C.

The stability of the structure of troponin C (calcium-binding components of troponin from rabbit skeletal muscle) has been studied by the scanning microcalorimetry method. It has been shows that: 1. In the presence of divalent ions the protein structure is represented by two practically independent cooperative blocks, one of which contains Ca2+-specific binding sites, and the other (Ca2+, Mg2+)-binding sites. 2. The stability of the cooperative block containing Ca2+-specific binding sites depends only on the concentration of Ca2+ and in its absence the melting temperature of the block decreases to 58 degrees C at neutral pH and low ionic strength. 3. The stability of the cooperative block containing (Ca2+, Mg2+)-binding sites depends on the concentration of Ca2+ or Mg2+. In their absence the stability of the block is so low that its structure is already disrupted at 25 degrees C. The conformational transition observed by different methods when divalent ions are removed is nothing else than the breaking down of the structure of this cooperative block.

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.