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T R Sosnick

Publications and source records attributed to T R Sosnick.

18 recordsLinked to original sources
PubMed1999

Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil.

We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state. Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c. We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants. Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited. The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces. These results are interpreted in relation to various models for protein folding.

Amino Acid Sequence↗
PubMed1998

Trifluoroethanol promotes helix formation by destabilizing backbone exposure: desolvation rather than native hydrogen bonding defines the kinetic pathway of dimeric coiled coil folding.

We measure the effects of low concentrations of helix-stabilizing cosolvents, including 2,2,2-trifluoroethanol (TFE), on the thermodynamics and kinetics of folding of the dimeric alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. The change in kinetic behavior upon addition of 5% (v/v) TFE indicates that it stabilizes the transition state to the same degree as the fully helical native state. However, folding rates are largely insensitive to alanine to glycine mutagenesis, indicating that the majority of helical structure is formed after the transition state. Equilibrium hydrogen isotope partitioning measurements indicate that intramolecular hydrogen bonds are not strengthened by TFE and that amide hydrogen bonds in the transition state are nearly the same strength as those in the unfolded state. Thus, the mechanism by which TFE exerts its helix-stabilizing effects can be divorced from helix formation and does not depend on the strengthening of intrahelical hydrogen bonds. Rather, TFE increases the structure of the binary alcohol/water solvent, thereby increasing the energetic cost associated with solvation of the polypeptide backbone. At low concentrations, TFE destabilizes the unfolded species and thereby indirectly enhances the kinetics and thermodynamics of folding of the coiled coil. A high degree of polypeptide backbone desolvation, and not the formation of regular helical structure and native strength hydrogen bonds, is the critical feature of the transition state for folding of this small dimeric protein.

Amino Acid Sequence↗
PubMed1998

The burst phase in ribonuclease A folding and solvent dependence of the unfolded state.

Submillisecond burst phase signals measured in kinetic protein folding experiments have been widely interpreted in terms of the fast formation of productive folding intermediates. Experimental comparisons with non-folding polypeptide chains show that, for ribonuclease A and cytochrome c, these signals in fact reflect a shift from one biased ensemble of the unfolded state to another as a function of change in denaturant concentration.

Animals↗
PubMed1997

Ultrafast signals in protein folding and the polypeptide contracted state.

To test the significance of ultrafast protein folding signals (<<1 msec), we studied cytochrome c (Cyt c) and two Cyt c fragments with major C-terminal segments deleted. The fragments remain unfolded under all conditions and so could be used to define the unfolded baselines for protein fluorescence and circular dichroism (CD) as a function of denaturant concentration. When diluted from high to low denaturant in kinetic folding experiments, the fragments readjust to their new baseline values in a "burst phase" within the mixing dead time. The fragment burst phase reflects a contraction of the polypeptide from a more extended unfolded condition at high denaturant to a more contracted unfolded condition in the poorer, low denaturant solvent. Holo Cyt c exhibits fluorescence and CD burst phase signals that are essentially identical to the fragment signals over the whole range of final denaturant concentrations, evidently reflecting the same solvent-dependent, relatively nonspecific contraction and not the formation of a specific folding intermediate. The significance of fast folding signals in Cyt c and other proteins is discussed in relation to the hypothesis of an initial rate-limiting search-nucleation-collapse step in protein folding [Sosnick, T. R., Mayne, L. & Englander, S. W. (1996) Proteins Struct. Funct. Genet. 24, 413-426].

Animals↗
PubMed1997

Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends.

The beta-thymosins are a family of highly polar peptides which serve in vivo to maintain a reservoir of unpolymerized actin monomers. In vitro, beta-thymosins form 1:1 complexes with actin monomers and inhibit both polymerization and exchange of the bound nucleotide. Circular dichroism data indicate that free thymosin beta 4 is predominantly unstructured, containing at most six residues of alpha-helix, and that up to six additional residues may adopt an alpha-helical conformation upon binding actin. NMR data indicate that many parts of thymosin beta 4 are not in tight contact with actin. Contacts between specific residues in actin and thymosin beta 4 were identified by zero-length cross-linking followed by isolation and sequencing of cross-linked peptides. After carbodiimide-mediated cross-linking, Lys-3 of thymosin beta 4 was cross-linked to Glu-167 of actin, and Lys-18 of thymosin beta 4 was cross-linked to one of the the N-terminal acidic residues of actin (Asp-1-Glu-4); the cross-linked actin residues lie within subdomains 3 and 1, respectively. These two contacts flank the alpha-helical region of thymosin beta 4 and place it on the barbed end; thymosin beta 4 can thus block actin polymerization sterically. After transglutaminase-mediated cross-linking, Lys-38 of thymosin beta 4 was cross-linked to Gln-41 of actin, placing the C-terminal region of thymosin beta 4 in contact with subdomain 2 on the pointed end; thymosin beta 4 may sterically block actin polymerization at the pointed end as well as the barbed end of the monomer. The distance between the pointed-end and barbed-end contacts requires that the C-terminal half of thymosin beta 4 be in a predominantly extended conformation.

Actins↗
PubMed1997

Hydrogen exchange: the modern legacy of Linderstrøm-Lang.

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm-Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.

Chemistry↗
PubMed1997

Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity.

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.

Bacillus subtilis↗
PubMed1996

Molecular collapse: the rate-limiting step in two-state cytochrome c folding.

Experiments with cytochrome c (cyt c) show that an initial folding event, molecular collapse, is not an energetically downhill continuum as commonly presumed but represents a large-scale, time-consuming, cooperative barrier-crossing process. In the absence of later misfold-reorganization barriers, the early collapse barrier limits cyt c folding to a time scale of milliseconds. The collapse process itself appears to be limited by an uphill search for some coarsely determined transition state structure that can nucleate subsequent energetically downhill folding events. An earlier "burst phase" event at strongly native conditions appears to be a non-specific response of the unfolded chain to reduced denaturant concentration. The molecular collapse process may or may not require the co-formation of the amino- and carboxyl-terminal helices, which are present in an initial metastable intermediate directly following the rate-limiting collapse. After the collapse-nucleation event, folding can proceed rapidly in an apparent two-state manner, probably by way of a predetermined sequence of metastable intermediates that leads to the native protein structure (Bai et al., Science 269:192-197, 1995).

Cytochrome c Group↗
PubMed1996

The role of helix formation in the folding of a fully alpha-helical coiled coil.

To determine when secondary structure forms as two chains coalesce to form an alpha-helical dimer, the folding rates of variants of the coiled coil region of GCN4 were compared. Residues at non-perturbing positions along the exterior length of the helices were substituted one at a time with alanine and glycine to vary helix propensity and therefore dimer stability. For all variants, the bimolecular folding rate remains largely unchanged; the unfolding rate changes to largely account for the change in stability. Thus, contrary to most folding models, widespread helix is not yet formed at the rate-limiting step in the folding pathway. The high-energy transition state is a collapsed form that contains little if any secondary structure, as suggested for the globular protein cytochrome c (Sosnick et al., Proteins 24: 413-426, 1996).

Amino Acid Sequence↗
PubMed1996

Mechanisms and uses of hydrogen exchange.

Recent work has largely completed our understanding of the hydrogen-exchange chemistry of unstructured proteins and nucleic acids. Some of the high-energy structural fluctuations that determine the hydrogen-exchange behavior of native macromolecules have been explained; others remain elusive. A growing number of applications are exploiting hydrogen-exchange behavior to study difficult molecular systems and elicit otherwise inaccessible information on protein structure, dynamics and energetics.

Humans↗
PubMed1995

Protein folding intermediates: native-state hydrogen exchange.

The hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.

Cytochrome c Group↗
PubMed1995

Structures of fd gene 5 protein.nucleic acid complexes: a combined solution scattering and electron microscopy study.

Small-angle scattering and electron microscopy studies of fd gene 5 protein (g5p) and reconstituted g5p.nucleic acid complexes have been used to test models for the complexes and evaluate their uniqueness. In addition, we have obtained new information on the dependence of nucleotide type and protein/nucleotide (P/N) ratio on the structure of the complexes. Reconstituted complexes were made with single-stranded fd viral DNA (fd ssDNA), poly[d(A)] and poly[r(A)]. All complexes form similar left-handed, flexible superhelices having approximately the same diameter, but the pitch differs among these complexes. The g5p protein is a dimer in solution and the dimers associate to form a superhelical framework to which the polynucleotide is attached. The combined X-ray and neutron scattering data confirm the nucleic acid is inside the protein superhelix. A Monte Carlo integration modeling procedure applied to the scattering data was used to systematically test large numbers of possible models for each complex, and previously proposed models based on parameters obtained from electron microscopy were found to be essentially correct and unique. The data on the complexes with different P/N ratios showed that mass per unit length values decreased while the rise per dimer and pitch of the superhelix increased for g5p.fd-ssDNA complexes with decreasing P/N ratios.

DNA, Single-Stranded↗
PubMed1994

The barriers in protein folding.

Elimination of an interaction which forms in denatured cytochrome c enables the majority of the molecules to fold to the native state on a 15 ms time scale, without populating observable intermediates. These results are contrary to the current view that particular steps in protein folding, including the supposedly rate-limiting molten globule to native transition, are intrinsically slow. Instead it appears that intermediates characterized so far may be kinetically trapped by barriers that are optional rather than integral to the folding process. Major barriers may result from misorganization of the chain in the initial condensation step.

Animals↗
PubMed1993

Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide.

Small-angle X-ray scattering and Fourier transform infrared (FTIR) spectroscopy experiments have been completed on the catalytic subunit of the cAMP-dependent protein kinase. Measurements were made both with and without the protein kinase inhibitor peptide, PKI alpha(5-22)amide. Binding of the peptide results in an overall contraction of the structure that is characterized by a decrease of 9% in radius of gyration and about 16% in the maximum linear dimension. Both the secondary structure content of the protein/peptide complex, as determined by FTIR, and the solution structure of this binary complex, as determined by X-ray scattering, agree well with the structural characteristics of this complex as elucidated by the crystal structure [Knighton, D.R., Zheng, J., Ten Eyck, L. F., Ashford, V.A., Xuong, N.H., Taylor, S.S., & Sowadsi, J. M. (1991a) Science 253, 407-414]. Further, the contraction of the structure observed by X-ray scattering upon inhibitor peptide binding is not accompanied by any detectable change in secondary structure content of the kinase. We have modeled the contraction of the kinase upon inhibitor peptide binding as a simple rotation of the large and small lobes seen in the crystal structure such that the cleft between them is closed. For a substrate these changes would then allow catalysis to ensue. The hinge for this movement occurs around a glycine that is one of the protein kinase family consensus amino acids.

Amino Acid Sequence↗
PubMed1992

Denatured states of ribonuclease A have compact dimensions and residual secondary structure.

Using small-angle X-ray scattering and Fourier transform infrared spectroscopy, we have determined that the thermally denatured state of native ribonuclease A is on average a compact structure having residual secondary structure. Under strongly reducing conditions, the protein further unfolds into a looser structure with larger dimensions but still retains a comparable amount of secondary structure. The dimensions of the thermally and chemically denatured states of the reduced protein are different but both are more compact than is predicted for a random coil of the same length. These results demonstrate that thermal denaturation in ribonuclease A is not a simple two-state transition from a native to a completely disordered random coil state.

Animals↗
PubMed1992

Distances between the antigen-binding sites of three murine antibody subclasses measured using neutron and X-ray scattering.

For three different murine immunoglobulins (IgG subclasses 1, 2a, and 2b), the distances between their antigen-binding sites have been measured using neutron scattering from deuterated antigens complexed with proteated IgG. Neutron-scattering data were measured for each antibody-antigen complex in a 41% D2O solvent. Unlike the proteated antibody molecule, the deuterated antigens are strongly contrasted against the 41% D2O solvent and give rise to a scattering profile that contains an interference term related to the distance between the deuterated antigens. For all three subclasses, the damping of this interference term, which gives information on the relative flexibility of the antigen-binding sites, indicates that a single distance is inadequate to describe the observed scattering and a distribution of distances is needed. The scattering profile has been modeled for each subclass to give the mean distance between the antigens and the variance of this distance. For all three IgG subclasses, the mean distance is between 117 and 134 A, and the variance is large (approximately 40 A), indicating a high degree of flexibility of the Fab arms. Small-angle X-ray scattering measurements on the same samples are consistent with the neutron-scattering results.

Animals↗