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S Marqusee

Publications and source records attributed to S Marqusee.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molten globule unfolding monitored by hydrogen exchange in urea.

The molten globule state of Escherichia coli ribonuclease H1 was studied by hydrogen exchange in order to understand how the energetics of individual regions react to the presence of denaturant. Hydrogen exchange rates were monitored (1) directly by NMR spectroscopy and (2) indirectly by quenching the exchange process and returning to the native state for NMR detection. Direct hydrogen exchange on the molten globule state demonstrated that the observed protons exchange 3-100-fold more slowly than in an unfolded peptide. The quenched hydrogen exchange experiments were modeled after the recently developed "native state hydrogen exchange" experiment and were carried out as a function of urea concentration. The free energy of hydrogen exchange varied linearly with denaturant concentration for all 29 measurable protons, suggesting that each amide's exchange behavior can be modeled with only one type of opening transition. The free energy of unfolding measured by hydrogen exchange and corresponding m values varied for each residue implying a noncooperative molten globule structure. These results are in contrast to similar exchange experiments on native proteins which generally display more than one type of exchange behavior. The single type of exchange seen in the molten globule is probably due to its larger conformational freedom and noncooperative nature.

Circular Dichroism↗
PubMed1998

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme.

The folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single-domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two-state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N-terminal subdomain (residues 13-75), and a C-terminal subdomain (residues 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full-length variants. We propose that this acid state is comprised of an unfolded N-terminal subdomain and a loosely folded C-terminal subdomain.

Anilino Naphthalenesulfonates↗
PubMed1998

Characterization of a folding intermediate from HIV-1 ribonuclease H.

The RNase H domain from HIV-1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped-flow far UV circular dichroism and pulse-labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog from Escherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C-terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV-1 therapy.

Circular Dichroism↗
PubMed1998

Hydrogen exchange studies of protein structure.

Hydrogen exchange techniques, with their residue-level specificity, exquisite sensitivity, and adaptability to many solution conditions, are becoming essential to the study of protein stability, folding and dynamics. Recent studies have elucidated the structures of intermediates formed transiently during protein folding and rare partially folded ensembles present at equilibrium. Analysis of hydrogen exchange mechanisms has revealed protein stability and kinetics at the level of individual residues.

Hydrogen↗
PubMed1997

Importance of the C-terminal helix to the stability and enzymatic activity of Escherichia coli ribonuclease H.

The ribonuclease H (RNase H) family of enzymes selectively degrades the RNA strand of RNA-DNA hybrids. This activity is essential for retroviruses such as HIV and resides in a domain of the larger reverse transcriptase molecule. RNase H from Escherichia coli is the best-characterized member of the family and serves as a model for structure/function studies. Despite having almost identical alpha + beta folds, the isolated domain from HIV is inactive and much less stable than the E. coli homolog. The HIV domain also shows increased disorder in its C-terminal regions (E-helix and His-containing loop). We investigated the importance of this region by studying a variant of E. coli RNase H lacking these elements (RNHdeltaE). Despite the elimination of 33 of 155 residues (including a complete helix), this C-terminal deletion mutant folds cooperatively as a subdomain. Surprisingly, this protein lacking residues near the active site retains weak Mn2+-dependent activity. A peptide corresponding to the deleted E-helix is helical in isolation and stimulates the activity of the deletion mutant in vitro. These results have implications for the catalytic mechanism of RNase H and drug design targeted to HIV reverse transcriptase.

Amino Acid Sequence↗
PubMed1997

The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions.

Folding of ribonuclease HI from Escherichia coli populates a kinetic intermediate detectable by stopped-flow circular dichroism. Pulse labelling hydrogen exchange reveals that this intermediate consists of a structured core region of the protein, namely helices A and D and beta-strand 4. This kinetic intermediate resembles both the acid molten globule of ribonuclease HI and rarely populated, partially unfolded forms detected under native conditions. These results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.

Circular Dichroism↗
PubMed1996

Structure of the acid state of Escherichia coli ribonuclease HI.

Under acidic conditions Escherichia coli ribonuclease HI* (RNase H*) adopts a partially folded state with all of the properties of a molten globule. Using amide hydrogen exchange carried out under acid state conditions, followed by quenching and NMR detection on the native state, we have determined the residues that are responsible for the observed structure of the acid state. Although RNase H* is a mixed alpha + beta protein, a helical subdomain (helices A, D, and B) defines the structure of the acid state. This structure correlates with the rare higher energy conformations detected under native conditions and with data for the earliest intermediates populated in the kinetic folding pathway of the protein.

Amino Acid Sequence↗
PubMed1996

The putative substrate recognition loop of Escherichia coli ribonuclease H is not essential for activity.

The RNase H family of enzymes catalyzes the hydrolysis of RNA from RNA DNA hybrids in a divalent metal-dependent fashion. To date, structure/function studies have focused on two members of this family: Escherichia coli RNase HI, a small monomeric protein; and human immunodeficiency virus, type I (HIV) RNase H, a domain of HIV reverse transcriptase. The isolated RNase H domain from HIV reverse transcriptase can be expressed independently and shares significant structural homology with its E. coli homologue; however, unlike the bacterial protein, it is inactive. The most notable difference between the inactive domain from HIV and the active E. coli protein is a basic helix/loop sequence, present in E. coli but absent from the HIV homologue. Substitution of this basic region into the HIV domain partially restores its activity and increases its thermodynamic stability. By deleting the basic helix/loop region, we have modeled the structural difference between these two polypeptides onto the E. coli homologue. Surprisingly, the resulting mutant protein is active in Mn2+-dependent fashion. Therefore, the basic helix/loop is not required for RNase H activity.

Amino Acid Sequence↗
PubMed1996

Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH.

Despite the general observation that single domain proteins denature in a completely cooperative manner, amide hydrogen exchange of ribonuclease H in low levels of denaturant demonstrates the existence of two partially folded species. The structures of these marginally stable species resemble kinetic folding intermediates and the molten globule state of the protein. These data suggest that the first region to fold is the thermodynamically most stable portion of the protein and that the molten globule is a high free energy conformation present at equilibrium in the native state.

Amides↗
PubMed1995

Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a multifunctional protein, containing both DNA polymerase and RNase H activity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RNA strand of RNA.DNA hybrids. While the domain that carries out the RNase H activity in HIV RT can be expressed as an independent, folded polypeptide, it is inactive as an RNase H. Here, we report the overexpression and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 basic helix/loop has been substituted for the corresponding sequence of HIV RNase H. The resulting polypeptide (RNH102) has Mn(2+)-dependent RNase H activity and is more stable than the independently expressed wild-type HIV RNase H domain.

Amino Acid Sequence↗
PubMed1994

Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state.

We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2-state process. Acid denaturation, however, reveals a cooperative transition at approximately pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric. Although compact, the acid state does not resemble the native protein: the acid state displays a near-UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1-anilinonaphthalene, 8-sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.

Circular Dichroism↗
PubMed1994

Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor.

In the N-terminal domain of lambda repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild-type N-terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side-chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild-type side chain on protein stability have been estimated. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure.

Amino Acid Sequence↗
PubMed1991

Calorimetric determination of the enthalpy change for the alpha-helix to coil transition of an alanine peptide in water.

The enthalpy change (delta H) accompanying the alpha-helix to random coil transition in water has been determined calorimetrically for a 50-residue peptide of defined sequence that contains primarily alanine. The enthalpy of helix formation is one of the basic parameters needed to predict thermal unfolding curves for peptide helices and it provides a starting point for analysis of the peptide hydrogen bond. The experimental uncertainty in delta H reflects the fact that the transition curve is too broad to measure in its entirety, which precludes fitting the baselines directly. A lower limit for delta H of unfolding, 0.9 kcal/mol per residue, is given by assuming that the change in heat capacity (delta Cp) is zero, and allowing the baseline to intersect the transition curve at the lowest measured Cp value. Use of the van't Hoff equation plus least-squares fitting to determine a more probable baseline gives delta H = 1.3 kcal/mol per residue. Earlier studies of poly(L-lysine) and poly(L-glutamate) have given 1.1 kcal/mol per residue. Those investigations, along with our present result, suggest that the side chain has little effect on delta H. The possibility that the peptide hydrogen bond shows a correspondingly large delta H, and the implications for protein stability, are discussed.

Alanine↗
PubMed1990

Relative helix-forming tendencies of nonpolar amino acids.

An important issue in understanding the relationship between protein sequence and structure is the degree to which different amino acids favour the formation of particular types of secondary structure. Estimates of the 'helix-forming tendency' of amino acids have been made based on 'host-guest' experiments, in which copolymers are made of the amino acid of interest (the 'guest') and a host residue (typically hydroxypropyl- or hydroxybutyl-L-glutamine). Recently, however, short alanine-based peptides were found to form stable monomeric helices in water, contrary to the result predicted from host-guest experiments. We have now measured the helix-forming tendency of five different nonpolar amino acids (Ala, Ile, Leu, Phe, Val) by substituting each in turn for alanine in a 17-residue alanine-based peptide and determining the extent of alpha-helix formation. Our results differ from those of host-guest experiments both in the degree of variation in helix-forming tendency of different amino acids, and in the rank order of the helix-forming tendency. We conclude that the helix-forming tendency of a particular amino acid depends on the sequence context in which it occurs; and the restriction of side-chain rotamer conformations is important in determining the helix-forming tendency.

Alanine↗
PubMed1989

Unusually stable helix formation in short alanine-based peptides.

Short, 16-residue, alanine-based peptides show stable alpha-helix formation in H2O. This result is surprising when contrasted with the classical view that regards the alpha-helix as a marginally stable structure in H2O and considers short helices unstable. The alanine-based peptides are solubilized by insertion of three or more residues of a single charge type, lysine (+) or glutamic acid (-). The results cannot be explained by helix stabilization resulting from concentration-dependent association or by the interaction of charged residues with the helix dipole. Our results are not predicted by the parameters for alanine and lysine that have been determined by the "host-guest" method: these parameters predict that a 16-residue peptide should not show measurable alpha-helix formation. Analysis of the role of the hydrophobic interaction in alpha-helix formation [Richards, F.M. & Richmond, T. (1978) in Molecular Interactions and Activity in Proteins, Ciba Foundation Symposium 60, ed. Wolstenholme, G.E. (Excepta Medica Amsterdam), pp. 23-25] does not show an unusually strong hydrophobic interaction in a helical block of alanine residues. The likely explanation for our results is, therefore, that individual alanine residues have a high helical potential. It is not yet known whether any other amino acids show this property, and the origin of this property is also unknown.

Alanine↗
PubMed1987

Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design.

Four alanine-based peptides were designed, synthesized, and tested by circular dichroism for alpha-helix formation in H2O. Each peptide has three glutamic/lysine residue pairs, is 16 or 17 amino acids long, and has blocked alpha-NH2 and alpha-COOH groups. In one set of peptides ("i+4"), the glutamic and lysine residues are spaced 4 residues or 1 residue apart. In the other set ("i+3"), the spacing is 3 or 2 residues. Within each of these sets, a pair of peptides was made in which the positions of the glutamic and lysine residues are reversed [Glu, Lys (E,K) vs. Lys, Glu (K,E)] in order to assess the interaction of the charged side chains with the helix dipole. Since the amino acid compositions of these peptides differ at most by a single alanine residue, differences in helicity are caused chiefly by the spacing and positions of the charged residues. The basic aim of this study was to test for helix stabilization by (Glu-, Lys+) ion pairs or salt bridges (H-bonded ion pairs). The results are as follows. (i) All four peptides show significant helix formation, and the stability of the alpha-helix does not depend on peptide concentration in the range studied. The best helix-former is (i+4)E,K, which shows approximately 80% helicity in 0.01 M NaCl at pH 7 and 0 degree C. (ii) The two i+4 peptides show more helix formation than the i+3 peptides. pH titration gives no evidence for helix stabilization by i+3 ion pairs. (iii) Surprisingly, the i+4 peptides form more stable helices than the i+3 peptides at extremes of pH (pH 2 and pH 12) as well as at pH 7. These results may be explained by helix stabilization through Glu-...Lys+ salt bridges at pH 7 and singly charged H bonds at pH 2 (Glu0...Lys+) and pH 12 (Glu-...Lys0). The reason why these links stabilize the alpha-helix more effectively in the i+4 than in the i+3 peptides is not known. (iv) Reversal of the positions of glutamic and lysine residues usually affects helix stability in the manner expected for interaction of these charged groups with the helix dipole. (v) alpha-Helix formation in these alanine-based peptides is enthalpy-driven, as is helix formation by the C-peptide of ribonuclease A.

Alanine↗