GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs.

The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system. The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile ... (mMFTI) or Met-Phe-Phe-Gly ... (mMFFG) or with poly(U). The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography. With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu.GTP)2.Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu.GTP.Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess. Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used. In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed. In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP. Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated. The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation.     

Folding of a large protein at high structural resolution

Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.Fifty years after Anfinsen’s seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3–7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8–12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work has demonstrated an advanced hydrogen-exchange pulse-labeling mass-spectrometry technology (HX MS) that is able to detect and characterize local structure, even when it is only transiently present during the course of kinetic folding (15, 16). The HX pulse-labeling approach provides a snapshot of main chain amide sites that are protected against HX labeling by H bonds present at the time of the labeling pulse (17, 18). HX MS measurements can determine the position, stability, and dynamic behavior of native and nonnative H-bonded structure and whether it persists or dissipates in subsequent folding. In a recent application, the method was able to describe the structure and time-dependent formation of three sequential native-like folding intermediates in the 155-residue ribonuclease H protein (15).Protein folding studies, whether theoretical or experimental, have been limited to relatively small proteins, with few exceptions. However, biological proteomes and the considerations they raise are dominated by large proteins (19). Here we extend the powerful HX MS technology to the two-domain, 370-residue, maltose binding protein (MBP). MBP is synthesized in the Escherichia coli cytoplasm and transported to the periplasm where it serves as a soluble receptor for the high-affinity capture and import of maltose and maltodextrins (20). The protein folds in vivo after deletion of a signal sequence (21); we study here the mature protein with the signal sequence deleted.When unfolded MBP is placed into native conditions, we find that it rapidly adopts a dynamic collapsed state, which can lead to aggregation in vitro when the concentration is >1 μM and to inclusion body formation in vivo (22). Folding to the native state occurs much more slowly even in the absence of aggregation, with all molecules moving through one or more intermediate states to the native state. The HX MS experiment provides incisive information on the nature of the initially collapsed state, the slow formation and identity of at least one on-pathway native-like intermediate, and the even slower emergence of native structure.     

The rate-limiting step in the folding of a large ribozyme without kinetic traps

A fundamental question in RNA folding is the nature of the rate-limiting step. Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates. The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps. We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering. Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates. Before the rate-limiting step, more than 98% of the total structure has formed. The rate-limiting step is a small-scale structural rearrangement involving prebound metal ions.