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J H Laity

Publications and source records attributed to J H Laity.

13 recordsLinked to original sources
PubMed2001

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A.

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.

Amino Acid Substitution↗
PubMed2001

Zinc finger proteins: new insights into structural and functional diversity.

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. Zinc finger structures are as diverse as their functions. Structures have recently been reported for many new zinc finger domains with novel topologies, providing important insights into structure/function relationships. In addition, new structural studies of proteins containing the classical Cys(2)His(2) zinc finger motif have led to novel insights into mechanisms of DNA binding and to a better understanding of their broader functions in transcriptional regulation.

Carrier Proteins↗
PubMed2000

Molecular basis for modulation of biological function by alternate splicing of the Wilms' tumor suppressor protein.

Alternate splicing, leading to the insertion of the tripeptide KTS in the linker between the third and fourth C(2)H(2) zinc fingers, changes both the DNA-binding function and the subnuclear localization of the Wilms' tumor suppressor protein (WT1). We have used NMR relaxation experiments to determine the molecular basis for the differing DNA recognition properties of the WT1-KTS and WT1+KTS isoforms. Our results show that the KTS insertion increases the flexibility of the linker between fingers 3 and 4 and abrogates binding of the fourth zinc finger to its cognate site in the DNA major groove. This represents a mechanism whereby a single zinc-finger gene can be used, through alternate splicing, to fulfill different functions in the cell.

Alternative Splicing↗
PubMed2000

Alternative splicing of Wilms' tumor suppressor protein modulates DNA binding activity through isoform-specific DNA-induced conformational changes.

The Wilms' tumor suppressor protein (WT1) is a zinc finger transcription factor that appears to function differently according to the presence of a posttranscriptional modification that adds three amino acids into one of the linker regions between the zinc fingers. We have investigated the structural consequences of the insertion of the Lys-Thr-Ser (KTS) sequence by preparing recombinant protein constructs of the four zinc finger DNA-binding domain of WT1 corresponding to the two isoforms with (+KTS) and without (-KTS) the insertion, which is located in the linker region between the third and fourth zinc fingers. NMR resonance assignments were used to estimate the structural differences between the two isoforms both free in solution and in complex with a 14 base pair DNA duplex corresponding to the WT1 recognition element. The NMR spectra indicate that the two isoforms are nearly identical in structure in the absence of the DNA. Only the immediate region of the insertion showed any change in chemical shifts. Upon DNA binding, the NMR spectrum of each isoform changed to indicate greater structure formation in the linker regions. Significant differences were observed between the spectra of the DNA complexes of the +KTS and -KTS isoforms, with the -KTS construct forming a more stable complex, consistent with prior biochemical assays. The majority of the differences between the spectra of the two complexes occur in the immediate region of the insertion, which appears to be closer in structure to the free form of the protein in the case of the +KTS complex. The insertion of the KTS sequence disrupts important interactions of the linker region with the adjacent zinc fingers, thus lowering the stability of the complex. The "normal" (-KTS) sequence of the linker appears to be involved in a C-terminal helix-capping interaction with the helix of the preceding zinc finger, a stabilizing interaction which is abrogated in the +KTS isoform.

Alternative Splicing↗
PubMed2000

DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers.

High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger proteins is mediated by both specific protein-base interactions and non-specific contacts between charged side-chains and the phosphate backbone. In addition, in DNA complexes of multiple zinc fingers, protein-protein interactions between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high- affinity binding, a highly specific DNA-induced helix capping involving residues in the linker sequence between fingers. Capping at the C terminus of the alpha-helix in each zinc finger, incorporating a consensus TGEKP linker sequence that follows each finger, provides substantial binding energy to the DNA complexes of zinc fingers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structures of each of the TGEKP linkers in these complexes can be superimposed on the linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structures from the zinc-finger-DNA complexes have been compared to the NMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 in the absence of DNA. This comparison reveals that additional stabilization likely arises in the DNA complexes from hydrogen bonding between the backbone amide of E3 and the side-chain O(gamma) of T1 in the linker. We suggest that these DNA-induced C-capping interactions provide a means whereby the multiple-finger complex, which must necessarily be domain-flexible in the unbound state as it searches for the correct DNA sequence, can be "snap-locked" in place once the correct DNA sequence is encountered. These observations provide a rationale for the high conservation of the TGEKP linker sequences in Cys(2)-His(2) zinc finger proteins.

Amino Acid Sequence↗
PubMed1999

Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway.

We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).

Amides↗
PubMed1998

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A.

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.

Animals↗
PubMed1997

Structural characterization of an analog of the major rate-determining disulfide folding intermediate of bovine pancreatic ribonuclease A.

The major rate-determining step in the oxidative regeneration of bovine pancreatic ribonuclease A (RNase A) proceeds through des-[40-95] RNase A, a three-disulfide intermediate lacking the Cys40-Cys95 disulfide bond. An analog of this intermediate, [C40A, C95A] RNase A, has been characterized in terms of regular backbone structure and thermodynamic stability at pH 4.6. Nearly complete backbone 1H, 15N, and 13C resonances, and most 13Cbeta side-chain resonances have been assigned for the mutant RNase A using triple-resonance NMR data and a computer program, AUTOASSIGN, for automated analysis of resonance assignments. Comparisons of chemical shift data, 3J(1HN-1Halpha) coupling constants, and NOE data for the mutant and wild-type proteins reveal that the overall chain folds of the two proteins are very similar, with localized structural perturbations in the regions spatially adjacent to the mutation sites in [C40A, C95A] RNase A. More significantly, 1H/2H amide exchange and thermodynamic data reveal a global destabilization of the mutant protein characterized by a significant difference in the midpoint of the thermal transition curves (DeltaTm of 21.8 degrees C) and a significant increase in the slowest exchanging backbone amide 1H/2H exchange rates (10(2)-10(6)-fold faster in the hydrophobic core of [C40A, C95 A] RNase A). Comparisons of the entropy DeltaS degrees (T) and enthalpy DeltaH degrees (T) of unfolding between wild-type and [C40A, C95A] RNase A reveal that some of the global destabilization of the mutant protein arises from entropic and enthalpic changes in the folded state. Implications of these observations for understanding the role of des-[40-95] in the folding pathway of RNase A are discussed.

Amino Acid Sequence↗
PubMed1997

Regeneration studies of an analog of ribonuclease A missing disulfide bonds 65-72 and 40-95.

Mutants of bovine pancreatic ribonuclease A (RNase A) that contain four of the eight cysteine residues found in the wild-type protein were prepared. Cysteine residues 40, 65, 72, and 95 were replaced by serine to form [C40S,C65S,C72S,C95S] RNase A or by alanine to form [C40A,C65A,C72A,C95A] RNase A, which contain the following four cysteine residues: 26, 58, 84, and 110. The substitutions resulted in deletion of wild-type disulfide bonds, 65-72 and 40-95. These mutants were prepared to investigate interactions that may be important for the folding and unfolding of the wild-type protein. The mutant protein was expressed and purified in an unfolded sulfonated form. Upon regeneration of the native form from the reduced mutant with DTTox, all three of the possible two-disulfide pairings, including the native one, formed. One-dimensional 1H NMR spectra demonstrated that the conformations of these three species are similar and are predominantly disordered; however, there is evidence of local structure in the vicinity of one histidine residue. It was also shown that disulfide pairing is not completely random and that both entropic factors and enthalpic interactions contribute to the formation of the native-disulfide bonds. The presence of more than a statistical population of native-disulfide pairings indicates that specific local interactions present in the reduced protein direct the preferential formation of native-disulfide bonds in the two-disulfide mutant.

Animals↗
PubMed1997

NMR structural analysis of an analog of an intermediate formed in the rate-determining step of one pathway in the oxidative folding of bovine pancreatic ribonuclease A: automated analysis of 1H, 13C, and 15N resonance assignments for wild-type and [C65S, C72S] mutant forms.

A three-disulfide intermediate, des-[65-72] RNase A, lacking the disulfide bond between Cys65 and Cys72, is formed in one of the rate-determining steps of the oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A). An analog of this intermediate, [C65S, C72S] RNase A, has been characterized in terms of structure and thermodynamic stability. Triple-resonance NMR data were analyzed using an automated assignment program, AUTOASSIGN. Nearly all backbone 1H, 13C, and 15N resonances and most side-chain 13C(beta) resonances of both wild-type (wt) and [C65S, C72S] RNase A were assigned unambiguously. Analysis of NOE, 13C(alpha) chemical shift, and 3J(H(N)-H(alpha)) scalar coupling data indicates that the regular backbone structure of the major form of [C65S, C72S] RNase A is very similar to that of the major form of wt RNase A, although small structural differences are indicated in the mutation site and in spatially adjacent beta-sheet structures comprising the hydrophobic core. Thermodynamic analysis demonstrates that [C65S, C72S] RNase A (Tm of 38.5 degrees C) is significantly less stable than wt RNase A (Tm of 55.5 degrees C) at pH 4.6. Although the structural comparison of wt RNase A and this analog of an oxidative folding intermediate indicates only localized effects around the Cys65 and Cys72 sites, these thermodynamic measurements indicate that formation of the fourth disulfide bond, Cys65-Cys72, on this oxidative folding pathway results in global stabilization of the native chain fold. This conclusion is supported by comparisons of amide 1H/2H exchange rates which are significantly faster throughout the entire structure of [C65S, C72S] RNase A than in wt RNase A. More generally, our study indicates that the C65-C72 disulfide bond of RNase A contributes significantly in stabilizing the structure of the hydrophobic core of the native protein. Formation of this disulfide bond in the final step of this oxidative folding pathway provides significant stabilization of the native-like structure that is present in the corresponding three-disulfide folding intermediate.

Amino Acid Sequence↗
PubMed1994

Folding pathway of guanidine-denatured disulfide-intact wild-type and mutant bovine pancreatic ribonuclease A.

The refolding kinetics of guanidine-denatured disulfide-intact bovine pancreatic ribonuclease A (RNase A) and its proline-42-to-alanine mutant (Pro42Ala) have been studied by monitoring tyrosine burial and 2'-cytidine monophosphate (2'CMP) inhibitor binding. The folding rate for wild-type RNase A is faster in the presence of the inhibitor 2'CMP than in its absence, indicating that the transition-state structure in the rate-determining step is stabilized by 2'CMP. The folding rate monitored by 2'CMP binding to the major slow-folding species of Pro42Ala RNase A is faster than the folding rate monitored by tyrosine burial; however, the folding rate monitored by inhibitor binding to the minor slow-folding species is decreased significantly over the folding rate monitored by tyrosine burial, indicating that the major and minor slow-folding species of Pro42Ala fold to the native state with different transition-state conformations in the rate-determining step.

Alanine↗
PubMed1993

Expression of wild-type and mutant bovine pancreatic ribonuclease A in Escherichia coli.

Wild-type ribonuclease A and five mutants thereof have been expressed in Escherichia coli as fusion proteins by using a T7 expression system. The five mutants are C[65-72]S, C[40-95]S, C[58-110]S, C[26-84]S, and K41G. The expressed fusion protein formed inclusion bodies which were then cleaved by factor Xa. The cleaved ribonuclease A was isolated as unfolded (sulfonated), soluble protein which was subsequently folded. This expression system can be used to produce mutants of ribonuclease A in yields suitable for folding and structural studies. All four native three-disulfide mutants exhibited enzymatic activity (5-30%), although only two were thermally stable at room temperature, demonstrating that no single native disulfide bond is essential for folding. The K41G mutant was enzymatically inactive with cyclic cytidine monophosphate as substrate.

Amino Acid Sequence↗