Biomedical subjects

P K Hammen

Publications and source records attributed to P K Hammen.

11 recordsLinked to original sources

PubMed2000

Structure of the cytosolic domain of TOM5, a mitochondrial import protein.

TOM5 is a small outer mitochondrial membrane protein in Saccharomyces cerevisiae and is part of a multi-protein translocator complex, which mediates protein import into mitochondria. Presently, nothing is known about the conformational preferences of TOM5 or other mitochondrial import proteins. In this report, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy are used to determine the conformational preferences of the cytosolic domain of TOM5. The CD spectra show evidence of a helical structure that is invariant with pH. NOESY data revealed that TOM5 forms a stable helical core between E11 and R15 with a less structurally rigid helix extending to the C-terminus.

Amino Acid Sequence↗

PubMed1999

The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N-terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria.

An apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one-fourth as well. It was shown that the N-terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N-terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co-translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of an Arg-Gly-Pro helix linker segment (residues 11-14) from the L2V mutant, which shifted three leucine residues toward the N-terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N-termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.

Aldehyde Dehydrogenase↗

PubMed1996

The role of positive charges and structural segments in the presequence of rat liver aldehyde dehydrogenase in import into mitochondria.

Most mitochondrial proteins are nucleus-encoded and translated in the cytosol. They have an N-terminal presequence that allows recognition by the mitochondrial import apparatus and subsequent import into mitochondria. These presequences are rich in positive charges, mainly arginines. The role of these positive charges in the 19-amino acid presequence of rat liver aldehyde dehydrogenase was investigated by systematically replacing them with the polar but uncharged residue, glutamine. The single substitution of any of the four Arg residues in the helical segments did not affect import. Substitution of both Arg residues in the N-terminal segment (R3Q/R10Q) caused a dramatic decrease in import competence. This could be restored by using the mutant lacking the three-amino acid (RGP) linker that separates the two helical domains, determined by two-dimensional NMR (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). CD and NMR spectra of the peptide corresponding to the linker-deleted presequence showed that it was substantially more prone to helix formation than the native peptide over its entire length. A similar analysis of the peptide corresponding to the R3Q/R10Q presequence revealed that this peptide was only somewhat more helical than the native peptide and that the greater helicity did not include the residues near the N terminus. It is concluded that positively charged residues in the presequence play a vital role in the import of precursor aldehyde dehydrogenase. One of the positive charges in the N-terminal helical segment of the presequence is necessary for import competence. However, if both positive charges are removed, import competence can be retained as long as the presequence is capable of forming a relatively more stable alpha-helix near its N terminus.

Aldehyde Dehydrogenase↗

PubMed1996

Influence of the mature portion of a precursor protein on the mitochondrial signal sequence.

Most mitochondrial proteins are synthesized with an N-terminal signal sequence that targets these proteins to various compartments within the mitochondria. Signal sequences have been shown to be functional by fusing them to a nonmitochondrial passenger protein and observing import. In many cases, a signal sequence has been fused to passenger proteins, such as dihydrofolate reductase, and import occurred. There are, though, several unexplained instances in which a signal sequence was attached to a passenger protein and import was not observed. In this study, the N-terminal 23 residues of the matrix enzyme rhodanese could import several passenger proteins but were unable to import the mature form of mitochondrial aldehyde dehydrogenase (mALDH). However, if these same 23 residues were fused to the middle portion of mALDH, import was recovered, suggesting that the rhodanese signal sequence and N terminus of mALDH were incompatible for import. Circular dichroism data indicated that a peptide corresponding to the region of fusion between rhodanese and mALDH had less structure than corresponding peptides from imported fusion proteins, suggesting that mALDH may alter the helix in the rhodanese signal sequence, thus preventing import.

Aldehyde Dehydrogenase↗

PubMed1996

Amphiphilicity determines binding properties of three mitochondrial presequences to lipid surfaces.

The interactions of three peptides, which correspond to presequences that direct mitochondrial protein import, with model membrane systems were characterized using NMR, fluorescence, and circular dichroism spectroscopies. The positively charged peptides adopted an ordered secondary structure only when the negatively charged phospholipid, cardiolipin, was present in small unilamellar vesicles. Conversely, the peptides adopted an ordered secondary structure in the presence of micelles formed from both formally neutral and negatively charged detergents. The peptides had the same relative affinity for micelles and small unilamellar vesicles containing 20% cardiolipin. Amide proton exchange rates showed that the region of the helical structure which had the greatest hydrophobic moment interacted most readily with micelles. Therefore, it appears that a major determinant of binding to lipid surfaces is the ability of the peptide to attain the correct orientation of hydrophobic and hydrophilic groups. For the three peptides studied, affinity also correlated with the length of the helix, but not with hydrophobic surface area. In each case, the interacting segment of the peptide was toward the C-terminal end of the helix. Previous work has allowed us to postulate that the N-terminus of the presequence is vital for import [Wang, Y., & Weiner, H. (1993) J. Biol. Chem. 268, 4759-4765] and the C-terminal end is essential for membrane interaction [Karslake, C., Piotto, M., Pak, Y. K., Weiner, H., & Gorenstein, D. G. (1990) Biochemistry 29, 9872-9878]. On the basis of the data that are now available, it appears that the interaction with membrane surfaces may depend on the location of an amphiphilic region of the sequence that is near but not necessarily at the C-terminus.

Acetyl-CoA C-Acyltransferase↗

PubMed1995

Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1994

Structure of the signal sequences for two mitochondrial matrix proteins that are not proteolytically processed upon import.

The N-terminal sequences of rhodanese and 3-oxoacyl-CoA thiolase, two mitochondrial matrix proteins that are not proteolytically processed upon import, have been studied by NMR and CD spectroscopy. In aqueous trifluoroethanol, in the presence of micelles, and in the presence of small unilamellar vesicles (SUVs), these peptides form alpha-helical structures beginning near the N-terminus and extending, continuously, for at least three helical turns. This result is consistent with a previous finding that a mutant rat liver mitochondrial aldehyde dehydrogenase signal sequence we designed, which formed a continuous alpha-helix, could successfully direct protein import but was not proteolytically processed (Thornton, K., Wang, Y., Weiner, H., & Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). From these three examples, a model is developed which suggests that a mitochondrial signal sequence that has an N-terminal alpha-helix longer than 11 residues can take on the necessary conformation to be imported but cannot adopt the necessary conformation to be processed.

Acetyl-CoA C-Acetyltransferase↗

PubMed1993

Deamidation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, involves asparagine 38 (HPr-1) and asparagine 12 (HPr-2) in isoaspartyl acid formation.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

Amino Acid Sequence↗

PubMed1991

Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy.

Analysis of the histidine-containing protein (HPr) from Escherichia coli by two-dimensional homonuclear and heteronuclear nuclear magnetic resonance techniques has been performed, extending the work originally reported [Klevit, R. E., Drobny, G. D., & Waygood, E. B. (1986) Biochemistry 25, 7760-7769; Klevit, R. E., & Drobny, G. P. (1986) Biochemistry 25, 7770-7773; Klevit, R. E., & Waygood, E. B. (1986) Biochemistry 25, 7774-7781]. Two-dimensional homonuclear total coherence spectroscopy (TOCSY) allowed for more complete assignments of the side-chain spin systems than had been possible in the original studies. As well, two-dimensional 15N-1H heteronuclear spectroscopy was used to resolve a number of ambiguities present in the homonuclear spectra due to resonance redundancies. These analyses led to the correction of a number of resonance assignments that were made with the spectra that could be collected with the technology that existed 6 years ago. In addition, amide exchange rates and 3JNH coupling constants have been measured, extending the original analysis and yielding new structural information. All these data have been used to reexamine the folding topology of E. coli HPr. Structure calculations showed that the topology derived from the earlier NMR data, i.e., a four-stranded beta-sheet with three alpha-helices running along one side of the sheet, was essentially unchanged, although at the present level of analysis, a well-defined "helix B" could not be established with high confidence. In addition, the data reported here revealed the existence of two slowly-exchanging side-chain hydroxyl protons belonging to Ser31 and Thr59. Their behavior strongly suggests that these side chains are involved in hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Computer-aided conformational analysis based on NOESY signal intensities.

The basis for the development of a suite of programs that allow the user to determine motional features (the correlation time and the significance of segmental motion) and the optimum conditions for future experiments from a NOESY signal matrix is presented. This automated evaluation of NOESY data serves as the initial step of an iterative conformational analysis which uses the molecular model manipulation capabilities of modern graphics workstations. Incorporated in these programs is NOESYSIM, a calculation subroutine which uses a set of molecular coordinates (and a correlation time estimate) together with user entered experimental parameters (acquisition time, sweep width, mixing time and cycle repetition time) to generate an accurately calculated NOESY signal matrix reflecting those conditions and the specified conformational model. Conformational refinement then consists of iterative comparisons of the experimental signal matrix with a series (or systematically sampled set) of model coordinates corresponding to a dynamics' course, driven-minimization or torsional grid search. These procedures and developments are illustrated with examples including: solution conformations of prostanoids; studies of the folding preferences and media-dependent changes in conformation for peptide hormones; and the structure elucidation of a novel undecapeptide macrolide antibiotic (lysobactin). For larger molecules, even constrained grid searches have too high a dimensionality and one must resort to distance-constraint based minimizations. A novel procedure for deriving more accurate distance constraints (corrected for secondary NOEs) is detailed and a new strategy for conformation elucidation, based on this procedure, is outlined.

Amino Acid Sequence↗

PubMedDate not supplied

Mitochondrial leader sequences: structural similarities and sequence differences.

While having essentially no amino acid sequence homology, the mitochondrial leader sequences of different pre-proteins carry out the same function of targeting the protein to mitochondria. Among the common attributes that have been noted for leader sequences are a net positive charge and the ability to form amphiphilic alpha-helices. The aim of the research described here was to determine the relative importance of these two attributes in the leader sequence of rat liver mitochondrial aldehyde dehydrogenase. Through site-directed mutagenesis, arginine residues were systematically replaced by glutamine. It was found that individual arginines could be replaced without loss of import competence, so the total charge of the leader sequence was not required for the pre-protein to be imported. However, when two arginines were replaced simultaneously, especially Arg3 and Arg10, the pre-protein lost the ability to be imported. This ability was restored by modifying the leader sequence to increase dramatically its helix-forming potential. This leader sequence did not contain a positive-charged side-chain until Arg11. Therefore, it has been shown that positive charge is not required in the first ten residues provided the sequence could form a relatively more stable alpha-helix. The ability to form an amphiphilic alpha-helix remains the essential factor in determining whether or not a leader sequence can carry out its import function.

Animals↗