Search PubMed⌕ Search

Biomedical subjects

L M Gierasch

Publications and source records attributed to L M Gierasch.

At least 19 recordsLinked to original sources
PubMed2001

Signal peptides bind and aggregate RNA. An alternative explanation for GTPase inhibition in the signal recognition particle.

N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle. In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of the Escherichia coli signal recognition particle, as previously reported (Miller, J. D., Bernstein, H. D., and Walter, P. (1994) Nature 367, 657-659), but not by the direct mechanism proposed. Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity. Surprisingly, only "functional" peptide sequences aggregate RNA; the peptides in general use as "nonfunctional" negative controls (e.g. those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle. We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in these in vitro studies and that signal sequences at the N terminus of nascent chains in vivo do not exhibit this activity.

GTP Phosphohydrolases↗
PubMed2001

Keeping it in the family: folding studies of related proteins.

Investigators have recently turned to studies of protein families to shed light on the mechanism of protein folding. In small proteins for which detailed analysis has been performed, recent studies show that transition-state structure is generally conserved. The number and structures of populated folding intermediates have been found to vary in homologous families of larger (greater than 100-residue) proteins, reflecting a balance of local and global interactions.

Amino Acid Sequence↗
PubMed2000

Multiple roles of prolyl residues in structure and folding.

To explore the ways that proline residues may influence the conformational options of a polypeptide backbone, we have characterized Pro-->Ala mutants of cellular retinoic acid-binding protein I (CRABP I). While all three Xaa-Pro bonds are in the trans conformation in the native protein and the equilibrium stability of each mutant is similar to that of the parent protein, each has distinct effects on folding and unfolding kinetics. The mutation of Pro105 does not alter the kinetics of folding of CRABP I, which indicates that the flexible loop containing this residue is passive in the folding process. By contrast, replacement of Pro85 by Ala abolishes the observable slow phase of folding, revealing that correct configuration of the 84-85 peptide bond is prerequisite to productive folding. Substitution of Pro39 by Ala yields a protein that folds and unfolds more slowly. Removal of the conformational constraint imposed by the proline ring likely raises the transition state barrier by increasing the entropic cost of narrowing the conformational ensemble. Additionally, the Pro-->Ala mutation removes a helix-termination signal that is important for efficient folding to the native state.

Circular Dichroism↗
PubMed2000

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding.

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.

Apoproteins↗
PubMed2000

Structural insights into substrate binding by the molecular chaperone DnaK.

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

Adenosine Triphosphatases↗
PubMed1999

Basis of substrate binding by the chaperonin GroEL.

The molecular chaperonins are essential proteins involved in protein folding, complex assembly, and polypeptide translocation. While there is abundant structural information about the machinery and the mechanistic details of its action are well studied, it is yet unresolved how chaperonins recognize a large number of structurally unrelated polypeptides in their unfolded or partially folded forms. To determine the nature of chaperonin-substrate recognition, we have characterized by NMR methods the interactions of GroEL with synthetic peptides that mimic segments of unfolded proteins. In previous work, we found using transferred nuclear Overhauser effect (trNOE) analysis that two 13 amino acid peptides bound GroEL in an amphipathic alpha-helical conformation. By extending the study to a variety of peptides with differing sequence motifs, we have observed that peptides can adopt conformations other than alpha-helix when bound to GroEL. Furthermore, peptides of the same composition exhibited significantly different affinities for GroEL as manifested by the magnitude of trNOEs. Binding to GroEL correlates well with the ability of the peptide to cluster hydrophobic residues on one face of the peptide, as determined by the retention time on reversed-phase (RP) HPLC. We conclude that the molecular basis of GroEL-substrate recognition is the presentation of a hydrophobic surface by an incompletely folded polypeptide and that many backbone conformations can be accommodated.

Amino Acid Sequence↗
PubMed1999

Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling.

In Escherichia coli, DnaK is essential for the replication of bacteriophage lambda DNA; this in vivo activity provides the basis of a screen for mutations affecting DnaK function. Mn PCR was used to introduce mutations into residues 405-468 of the C-terminal polypeptide-binding domain of DnaK. These mutant proteins were screened for the ability to propagate bacteriophage lambda in the background of a dnaK deficient cell line, BB1553. This initial screen identified several proteins which were mutant at multiple positions. The multiple mutants were further dissected into single mutants which remained negative for lambda propagation. Four of these single-site mutants were purified and assayed for biochemical functionality. Two single-site mutations, F426S and S427P, are localized in the peptide binding site and display weakened peptide binding affinity. This indicates that the crystallographically determined peptide binding site is also critical for in vivo lambda replication. Two other mutations, K414I and N451K, are located at the edge of the beta-sandwich domain near alpha-helix A. The K414I mutant binds peptide moderately well, yet displays defects in allosteric functions, including peptide-stimulated ATPase activity, ATP-induced changes in tryptophan fluorescence, ATP-induced peptide release, and elevated ATPase activity. The K414 position is close in tertiary structure to the linker region to the ATPase domain and reflects a specific area of the peptide-binding domain which is necessary for interdomain coupling. The mutant N451K displays defects in both peptide binding and allosteric interaction.

Adenosine Triphosphatases↗
PubMed1999

Unfolding dynamics of a beta-sheet protein studied by mass spectrometry.

The unfolding dynamics of cellular retinoic acid-binding protein I (CRABP I), an 18 kDa predominantly beta-sheet protein, were studied by monitoring the hydrogen-deuterium (H-D) exchange reaction under various solution conditions. A bimodal charge state distribution was observed when a denaturing agent was added to the protein aqueous solution. These two populations exhibit different kinetics of H-D exchange, with the high charge state ions undergoing very rapid isotope exchange, while the low charge state protein ions exchange cooperatively but at much slower rates. Transiently populated intermediate states were detected indirectly using hydrogen exchange measurement in aqueous solution at various pHs. At pH 2.5 and room temperature, three distinct populations of CRABP I ions exist over an extended period of time, each corresponding to a specific degree of backbone amide hydrogen atom protection. Mass spectral data are complementary to hydrogen exchange measurements by NMR, since the former samples a much faster time-scale of dynamic events in solution.

Deuterium↗
PubMed1998

The LDL receptor clustering motif interacts with the clathrin terminal domain in a reverse turn conformation.

Previously the hexapeptide motif FXNPXY807 in the cytoplasmic tail of the LDL receptor was shown to be essential for clustering in clathrin-coated pits. We used nuclear magnetic resonance line-broadening and transferred nuclear Overhauser effect measurements to identify the molecule in the clathrin lattice that interacts with this hexapeptide, and determined the structure of the bound motif. The wild-type peptide bound in a single conformation with a reverse turn at residues NPVY. Tyr807Ser, a peptide that harbors a mutation that disrupts receptor clustering, displayed markedly reduced interactions. Clustering motif peptides interacted with clathrin cages assembled in the presence or absence of AP2, with recombinant clathrin terminal domains, but not with clathrin hubs. The identification of terminal domains as the primary site of interaction for FXNPXY807 suggests that adaptor molecules are not required for receptor-mediated endocytosis of LDL, and that at least two different tyrosine-based internalization motifs exist for clustering receptors in coated pits.

Adaptor Protein Complex 2↗
PubMed1998

Molecular chaperones: clamps for the Clps?

The Clp/Hsp100 molecular chaperones are unusual in their ability to tease apart protein aggregates and complexes. Recent results make a good case that these chaperones bind substrates via PDZ-like domains; this may reflect a general strategy for manipulating the] assembly state of substrate proteins.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Probing the folding pathway of a beta-clam protein with single-tryptophan constructs.

BACKGROUND: Cellular retinoic acid binding protein I (CRABPI) is a small, predominantly beta-sheet protein with a simple architecture and no disulfides or cofactors. Folding of mutants containing only one of the three native tryptophans has been examined using stopped-flow fluorescence and circular dichroism at multiple wavelengths. RESULTS: Within 10 ms, the tryptophan fluorescence of all three mutants shows a blue shift, and stopped-flow circular dichroism shows significant secondary structure content. The local environment of Trp7, a completely buried residue located near the intersection of the N and C termini, develops on a 100 ms time scale. Spectral signatures of the other two tryptophan residues (87 and 109) become native-like in a 1 s kinetic phase. CONCLUSIONS: Formation of the native beta structure of CRABPI is initiated by rapid hydrophobic collapse, during which local segments of chain adopt significant secondary structure. Subsequently, transient yet specific interactions of amino acid residues restrict the arrangement of the chain topology and initiate long-range associations such as the docking of the N and C termini. The development of native tertiary environments, including the specific packing of the beta-sheet sidechains, occurs in a final, highly cooperative step simultaneous with stable interstrand hydrogen bonding.

Circular Dichroism↗
PubMed1997

Domain interactions in E. coli SRP: stabilization of M domain by RNA is required for effective signal sequence modulation of NG domain.

The E. coli protein, Fth, binds to 4.5S RNA through its M domain to form the signal recognition particle (SRP). The other domain of Fth (NG) is a GTPase, which binds and is coordinately regulated by its receptor, FtsY. We find that the helical M domain is inherently flexible. Binding of 4.5S RNA to Fth stabilizes the M domain yet has little apparent effect on the binding of signal peptides. However, in the absence of the RNA, signal peptide binding results in a global destabilization of Fth, which is prevented by binding of 4.5S RNA. Signal peptide binding to isolated NG domain also causes a pronounced destabilization, implicating the NG domain in direct recognition of signal peptide.

Amino Acid Sequence↗
PubMed1997

Local interactions in a Schellman motif dictate interhelical arrangement in a protein fragment.

BACKGROUND: As an approach to understanding the role of local sequence in determining protein tertiary structure, we have examined the conformation of a 23-residue peptide fragment corresponding to the structurally conserved helix-Schellman motif-helix (H-Sm-H) domain (residue 10-32) of cellular retinoic acid binding protein, along with variants designed to probe the contributions of the helix-terminating Gly23 and the hydrophobic interactions between Leu 19 and Val24 in stabilizing the Schellman motif and hence helix termination. RESULTS: In aqueous solution, NMR data for the H-Sm-H peptide show that it samples a largely helical conformation with a break in the helix at the point of the turn in the protein. The data also establish the presence of local hydrophobic interactions and intramolecular hydrogen bonds characteristic of a Schellman motif. Absence of helix termination in trifluoroethanol, a solvent known to disrupt hydrophobic interactions, along with an analysis of H alpha chemical shifts and NOEs in the variant peptides, suggest a major role for glycine in terminating the helix, with local hydrophobic interactions further stabilizing the Schellman motif. CONCLUSIONS: The presence of a Schellman motif in this isolated fragment in water is governed by local interactions and specifies the interspatial arrangement of the helices. This observation underlines the structure predictive value of folding motifs. As proposed for a Schellman motif, helix termination in this fragment is dictated by the local distribution of polar/apolar residues, which is reminiscent of the binary code for protein folding.

Amino Acid Sequence↗
PubMed1997

Cavity formation before stable hydrogen bonding in the folding of a beta-clam protein.

The time course of folding of a small beta-sheet protein reveals formation of a central ligand binding cavity before the consolidation of the native hydrogen bonding network. These results suggest that side chain interactions and not stable hydrogen bonding determine the beta-sheet architecture and play crucial roles in the overall chain topology.

Hydrogen Bonding↗
PubMed1996

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.

Circular Dichroism↗