Electrospray ionization mass spectrometry was used to investigate the structure of the Escherichia coli chaperone protein SecB. It was determined that the N-terminal methionine of SecB has been removed and that more than half of all SecB monomers are additionally modified, most likely by acetylation of the N-terminus or a lysine. The use of gentle mass spectrometer interface conditions showed that the predominant, oligomeric form of SecB is a tetramer that is stable over a range of solution pH conditions and mass spectrometer interface heating (i.e., inlet capillary temperatures). At very high pH, SecB dimers are observed. SecB contains a region that is hypersensitive to cleavage by proteinase K and is thought to be involved in conformational changes that are crucial to the function of SecB. We identified the primary site of cleavage to be between Leu 141 and Gln 142. Fourteen amino acids are removed, but the truncated form remains a tetramer with stability similar to that of the intact form.Keywords: chaperone; electrospray mass spectrometry; noncovalent interactions; SecB Molecular chaperones are found throughout nature participating in a wide range of processes, including folding, oligomerization, and subcellular localization of polypeptides (Randall & Hardy, 1995). All molecular chaperones selectively bind nonnative polypeptides with no affinity for proteins that have acquired their native state. The mechanism allowing chaperones to recognize a polypeptide as a ligand by virtue of the fact it is nonnative is of great interest. Although detailed structural information would be enormously valuable in understanding how a chaperone works, the structures of only a few chaperones have been determined at high resolution: GroEL (Braig et al., 1994) and Pap D (Holmgren & Braenden, 1989) by X-ray crystallography, and domains of Hsc 70 (Morshauser et al., 1995) and DnaJ (Szyperski et al., 1994) by NMR.We report here the first use of electrospray ionization mass spectrometry (ESI-MS) to investigate the quaternary structure of SecB, a chaperone protein of Escherichia coli. SecB is the only chaperone among the several identified in E. coli that is dedicated to the facilitation of protein export across the cytoplasmic membrane (Randall & Hardy, 1995 promoting the export of a subset of precursor proteins in vivo, SecB has also been shown to bind a number of proteins and peptides that possess nonnative structure even though they are not natural ligands found in E. coli. (Hardy & Randall, 1991). Efforts to crystallize SecB in several laboratories have not produced diffraction-quality crystals (Vrielink et al., 1995; Dodson, Guy G., pers. comm.), thus we have no information related to the 3D structure. SecB has been shown by CD to contain a high percentage of 6 structure, as well as regions with no defined secondary structure (Breukink et al., 1992;Fasman et al., 1995). The initial evidence that SecB is oligomeric came from analysis by size-exclusion chromatography and electrophoresis of the native protein through po...

SummaryIn all living cells regulated passage across membranes of specific proteins occurs through a universally conserved secretory channel. In bacteria and chloroplasts, the energy for the mechanical work of moving polypeptides through that channel is provided by SecA, a regulated ATPase. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to identify the interactive surface used by SecA for each of the diverse binding partners encountered during the dynamic cycle of export. Although the binding sites overlap, resolution at the level of aminoacyl sidechains allows us to identify contacts that are unique to each partner. Patterns of constraint and mobilization of residues on that interactive surface suggest a conformational change that may underlie the coupling of ATP hydrolysis to precursor translocation.

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The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods. Cleavage of the backbone after position 86 resulted in two fragments, {1-86} and {87-159}, each of which are poorly structured and enzymatically inactive. When combined in a 1 : 1 molar ratio, however, the fragments formed a high-affinity (K a ‫ס‬ 2.6 × 10 7 M −1) complex that displays a weakly cooperative urea-induced unfolding transition at micromolar concentrations. The retention of about 15% of the enzymatic activity of full-length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild-type counterpart. In contrast, a circularly permuted form with its N-terminus at position 86 has similar overall stability to full-length DHFR, about 50% of its activity, substantial secondary structure, altered side-chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild-type protein. After addition of ligand or the tightbinding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more nativelike secondary and tertiary structures. These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein-ligand interactions in stabilizing the active site architecture of DHFR.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochern Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.Keywords: analytical ultracentrifugation; calorimetry; chaperones; Fourier transform ion cyclotron resonance mass spectrometry; SecB SecB, a cytosolic tetrameric protein in Escherichia coli, is a chaperone that facilitates export of polypeptides to the periplasmic space and to the outer membrane (for review see Kumamoto, 1991;Collier, 1993;Hardy & Randall, 1993). SecB binds precursor proteins in the cytoplasm before they assume their native, stably folded structures and maintains them in a state that is compatible with transfer through the membrane. By binding proteins destined for export, SecB controls a kinetic partitioning between folding of the polypeptides to their native conformation in the cytoplasm, which is the wrong compartment, and export through the cytoplasmic membrane to their proper destination. Since folded precursors can neither bind SecB nor be exported, the proportion of the polypeptides that are properly localized is a function of the rate constant of folding relative to the rate constant of association with SecB (Randall & Hardy, 1995). The interaction of SecB with ligands is one of high affinity (Hardy & Randall, 1991) high rate constants for both association and dissociation (Fekkes et al., 1995;Randall & Hardy, 1995). Perhaps the most remarkable feature of this binding is that there is no apparent consensus in primary, secondary, or tertiary structure among the polypeptides that SecB has been shown to bind. Rather, the feature that distinguishes polypeptides as ligands for SecB is that they are in a nonnative conformation. A large part of the selectivity in vivo most likely results from the fact that precursors of exported proteins contain a leader sequence that slows the folding of the polypeptide thus poising the kinetic partitioning toward binding of SecB and thereby export. We have previously proposed a model based on studies with short peptide ligands to explain the ability...

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was camed out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1 : 1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.Keywords: dissociation; Fourier transform ion cyclotron resonance; MS/MS; OppA; protein export; SecB; stoichiometry Mass spectrometry has evolved to contribute to biological research on many frontiers (Ganem et al

Interactions between short protein segments and phospholipid bilayers dictate fundamental aspects of cellular activity and have important applications in biotechnology. Yet, the lack of a suitable methodology for directly probing these interactions has hindered the mechanistic understanding. We developed a precision atomic force microscopy-based single-molecule force spectroscopy assay and probed partitioning into lipid bilayers by measuring the mechanical force experienced by a peptide. Protein segments were constructed from the peripheral membrane protein SecA, a key ATPase in bacterial secretion. We focused on the first 10 amino-terminal residues of SecA (SecA2-11) that are lipophilic. In addition to the core SecA2-11 sequence, constructs with nearly identical chemical composition but with differing geometry were used: two copies of SecA2-11 linked in series and two copies SecA2-11 linked in parallel. Lipid bilayer partitioning interactions of peptides with differing structures were distinguished. To model the energetic landscape, a theory of diffusive barrier crossing was extended to incorporate a superposition of potential barriers with variable weights. Analysis revealed two dissociation pathways for the core SecA2-11 sequence with well-separated intrinsic dissociation rates. Molecular dynamics simulations showed that the three peptides had significant conformational differences in solution that correlated well with the measured variations in the propensity to partition into the bilayer. The methodology is generalizable and can be applied to other peptide and lipid species.

The general secretory (Sec) system of Escherichia coli translocates both periplasmic and outer membrane proteins through the cytoplasmic membrane. The pathway through the membrane is provided by a highly conserved translocon, which in E. coli comprises two heterotrimeric integral membrane complexes, SecY, SecE, and SecG (SecYEG), and SecD, SecF, and YajC (SecDF/YajC). SecA is an associated ATPase that is essential to the function of the Sec system. SecA plays two roles, it targets precursors to the translocon with the help of SecB and it provides energy via hydrolysis of ATP. SecA exists both free in the cytoplasm and integrally membrane associated. Here we describe details of association of the amino-terminal region of SecA with membrane. We use site-directed spin labelling and electron paramagnetic resonance spectroscopy to show that when SecA is co-assembled into lipids with SecYEG to yield highly active translocons, the N-terminal region of SecA penetrates the membrane and lies at the interface between the polar and the hydrophobic regions, parallel to the plane of the membrane at a depth of approximately 5 Å. When SecA is bound to SecYEG, preassembled into proteoliposomes, or nonspecifically bound to lipids in the absence of SecYEG, the N-terminal region penetrates more deeply (8 Å). Implications of partitioning of the SecA N-terminal region into lipids on the complex between SecB carrying a precursor and SecA are discussed.