Biomedical subjects

Stephen J Hagen

Publications and source records attributed to Stephen J Hagen.

13 recordsLinked to original sources

PubMed2007

Kinetics of internal-loop formation in polypeptide chains: a simulation study.

The speed of simple diffusional motions, such as the formation of loops in the polypeptide chain, places one physical limit on the speed of protein folding. Many experimental studies have explored the kinetics of formation of end-to-end loops in polypeptide chains; however, protein folding more often requires the formation of contacts between interior points on the chain. One expects that, for loops of fixed contour length, interior loops will form more slowly than end-to-end loops, owing to the additional excluded volume associated with the "tails". We estimate the magnitude of this effect by generating ensembles of randomly coiled, freely jointed chains, and then using the theory of Szabo, Schulten, and Schulten to calculate the corresponding contact formation rates for these ensembles. Adding just a few residues, to convert an end-to-end loop to an internal loop, sharply decreases the contact rate. Surprisingly, the relative change in rate increases for a longer loop; sufficiently long tails, however, actually reverse the effect and accelerate loop formation slightly. Our results show that excluded volume effects in real, full-length polypeptides may cause the rates of loop formation during folding to depart significantly from the values derived from recent loop-formation experiments on short peptides.

Computer Simulation↗

PubMed2006

Characterizing the residue level folding of the intrinsically unstructured IA3.

Residue level analysis of the folding of simple proteins may hold the key to understanding folding pathways and aid in structure prediction. IA(3), the endogenous inhibitor of yeast aspartic proteinase A (YPrA), is an unstructured protein in solution. Comparison of the 2D (15)N-HSQC spectra of IA(3) in water and in 23% 2,2,2-trifluoroethanol (TFE) shows that the individual residue cross peaks of IA(3) become more dispersed in the presence of TFE, indicating that the protein undergoes an unstructured to structured transition in the presence of TFE. This transition can be monitored by the movements of the cross peaks. Following the individual cross peaks, however, is complicated and does not establish whether a single transition occurs globally in the sequence. In this equilibrium study, we apply singular value decomposition (SVD) to elucidate both the main features of the TFE-driven transition and the residue-level deviations from the average behavior. This analysis has yielded a two-state folding description as well as specifics of NMR frequency shifts of individual residues, indicating that the N-terminus of IA(3) has a higher helical propensity than the C-terminus. Additionally, we discuss possible mechanisms for observed deviations from a two-state folding transition. When combined with a traditional biochemical understanding of interactions between individual residues, this approach leads to a better understanding of protein folding.

Aspartic Acid Endopeptidases↗

PubMed2006

Do protein molecules unfold in a simple shear flow?

Protein molecules typically unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although the protein literature contains many references to shear denaturation, we find little quantitative evidence for the phenomenon. We have investigated whether a high shear can destabilize a small globular protein to any measurable extent. We study a protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. By forcing the sample through a silica capillary (inner diameter 150-180 microm) at speeds approaching 10 m/s, we subject the protein to shear rates dv(z)/dr as large as approximately 2 x 10(5) s(-1) while illuminating it with an ultraviolet laser. We can readily detect fluorescence changes of <1%, corresponding to shifts of < approximately 0.01 kJ/mol in the stability of the folded state. We find no evidence that even our highest shear rates significantly destabilize the folded protein. A simple model suggests that extraordinary shear rates, approximately 10(7) s(-1), would be required to denature typical small, globular proteins in water.

Animals↗

PubMed2005

Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

Biochemistry↗

PubMed2004

Internal friction controls the speed of protein folding from a compact configuration.

Several studies have found millisecond protein folding reactions to be controlled by the viscosity of the solvent: Reducing the viscosity allows folding to accelerate. In the limit of very low solvent viscosity, however, one expects a different behavior. Internal interactions, occurring within the solvent-excluded interior of a compact molecule, should impose a solvent-independent upper limit to folding speed once the bulk diffusional motions become sufficiently rapid. Why has this not been observed? We have studied the effect of solvent viscosity on the folding of cytochrome c from a highly compact, late-stage intermediate configuration. Although the folding rate accelerates as the viscosity declines, it tends toward a finite limiting value approximately 10(5) s(-1) as the viscosity tends toward zero. This limiting rate is independent of the cosolutes used to adjust solvent friction. Therefore, interactions within the interior of a compact denatured polypeptide can limit the folding rate, but the limiting time scale is very fast. It is only observable when the solvent-controlled stages of folding are exceedingly rapid or else absent. Interestingly, we find a very strong temperature dependence in these "internal friction"-controlled dynamics, indicating a large energy scale for the interactions that govern reconfiguration within compact, near-native states of a protein.

Animals↗

PubMed2004

IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.

IA(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA(3) binds to YprA as an alpha-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113-117]. Surprisingly, only residues 2-32 of IA(3) are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.

Amino Acid Sequence↗

PubMed2004

A limiting speed for protein folding at low solvent viscosity.

Because protein folding dynamics are heavily overdamped, Kramers theory predicts the rate of folding to scale inversely with the reaction friction, which is usually interpreted to mean the solvent viscosity. This does not mean, however, that the speed of folding can increase without limit as solvent viscosity decreases. We show that, in a sufficiently fast-folding protein, the folding speed approaches a finite limit at low solvent viscosity, indicating a reaction controlled by internal friction.

Glucose↗

PubMed2004

Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy.

We studied the microsecond folding dynamics of three beta hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLS(aa)) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLS(aa) TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.

Computer Simulation↗

PubMed2003

Fast chain contraction during protein folding: "foldability" and collapse dynamics.

Theory indicates that at least some proteins will undergo a rapid and unimpeded collapse, like a disorganized hydrophobic chain, prior to folding. Yet experiments continue to find signs of an organized, or barrier-limited, collapse in even the fastest (approximately mus) folding proteins. Does the kinetic barrier represent a signature of the equilibrium "foldability" of these molecules? We have measured the rate of chain contraction in two nonfolding analogs of a very fast-collapsing protein. We find that these chains contract on the same time scale (approximately 10(-5)s) as the natural protein, and both pass over an energetic barrier at least as large as that encountered by the protein. The equilibrium foldability of the native structure therefore does not alone determine the dynamics of collapse; even the disordered chains contract approximately 1000x slower than expected for an ideal chain.

Animals↗

PubMed2003

Exponential decay kinetics in "downhill" protein folding.

The observation of single-exponential kinetic phases in early stages of protein folding is often interpreted as evidence that these phases are rate limited by significant energy or entropy barriers. However, although the existence of large barriers reliably implies exponential kinetics, the reverse is not necessarily true. A simple model for the hydrophobic collapse of a chain molecule demonstrates that a barrierless or "downhill" diffusional relaxation can give rise to kinetics that are practically indistinguishable from a pure exponential. Within this model, even a highly nonlinear experimental probe such as resonance energy transfer (Förster transfer) could exhibit a large amplitude decay (greater than 90% in fluorescence) that deviates from a simple exponential by less than 0.5%. Only a detailed analysis of the dynamics is likely to reveal that a free energy barrier is absent.

Fluorescence Resonance Energy Transfer↗

PubMed2002

Smaller and faster: the 20-residue Trp-cage protein folds in 4 micros.

We have used laser temperature jump spectroscopy to measure the folding speed of the 20-residue Trp-cage, the smallest polypeptide known to exhibit truly cooperative folding behavior. The observed folding time (4 mus at room temperature) makes this not only the smallest foldable protein, but also the fastest, with a folding speed that exceeds contact-order predictions and approaches anticipated diffusional "speed limits" for protein folding.

Amino Acid Sequence↗

PubMed2002

Rapid intrachain binding of histidine-26 and histidine-33 to heme in unfolded ferrocytochrome C.

Time-resolved spectroscopic studies of unfolded horse iron(II) cytochrome c have suggested that the imidazole side chains of His26 and His33 bind transiently to the heme iron on microsecond time scales, after photodissociation of a carbon monoxide ligand from the heme. Our studies of four variants of cytochrome c (horse wild type, horse H33N, horse H33N/H26Q, and tuna wild type), unfolded in guanidine hydrochloride at pH 6.5, demonstrate that these side chains are responsible for the observed microsecond spectral changes. As His33 and then His26 are eliminated from the horse wild-type sequence, transient optical absorption spectra show systematic suppression of a rapid (approximately 10-100 micros) Soret absorbance change that follows photolysis of CO. Transient binding of these histidine side chains to the heme therefore generates one of the fast kinetic phases observed in previous photochemically triggered spectroscopic studies of dynamics in unfolded iron(II) cytochrome c. Furthermore, both His33 and His26 appear to contribute to a similar extent in these early kinetics. Thus, the stiffness of the polypeptide chain creates a deviation from Gaussian chain behavior by impeding, although not preventing, the formation of short (<10 peptide bonds) intrachain loops around the heme group.

Animals↗

PubMed2002

Laminar-flow fluid mixer for fast fluorescence kinetics studies.

The ability to mix aqueous liquids on microsecond time scales, while consuming minimal amounts of sample and maintaining UV-visible optical access to the mixing region, is highly desirable for a range of biophysical studies of fast protein and nucleic acid interactions and folding. We have constructed a laminar coaxial jet mixer that allows the measurement of UV-excited fluorescence from nanoliter and microliter quantities of material, mixed at microsecond rates. The mixer injects a narrow cylindrical stream (radius a < 1 microm) of fluorescent sample into a larger flow of diluting buffer that moves through a capillary (100 microm i.d.) at a speed approximately 20 cm/s, under laminar flow conditions (Re approximately equal to 14). Construction from a fused silica capillary allows the laser excitation (at 266 nm) and detection (at 350 nm) of tryptophan fluorescence at reasonably low working concentrations, without interference from background fluorescence. Using this mixer we have measured sub-millisecond fluorescence quenching kinetics while consuming fluorescent sample at rates no greater than 6 nl/s. Consumption of the diluting buffer is also very modest (approximately 1-3 microl/s) in comparison with other rapid mixer designs.

Biophysics↗