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1.
Commun Chem ; 7(1): 81, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38600176

ABSTRACT

Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

2.
Appl Spectrosc ; 70(8): 1292-9, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27340218

ABSTRACT

A new method of performing pump-probe experiments is proposed and experimentally demonstrated by a proof of concept on the millisecond scale. The idea behind this method is to measure the total probe intensity arising from several time points as a group, instead of measuring each time separately. These measurements are multiplexes that are then transformed into the true signal via multiplication with a binary Hadamard S matrix. Each group of probe pulses is determined by using the pattern of a row of the Hadamard S matrix and the experiment is completed by rotating this pattern by one step for each sample excitation until the original pattern is again produced. Thus to measure n time points, n excitation events are needed and n probe patterns each taken from the n × n S matrix. The time resolution is determined by the shortest time between the probe pulses. In principle, this method could be used over all timescales, instead of the conventional pump-probe method which uses delay lines for picosecond and faster time resolution, or fast detectors and oscilloscopes on longer timescales. This new method is particularly suitable for situations where the probe intensity is weak and/or the detector is noisy. When the detector is noisy, there is in principle a signal to noise advantage over conventional pump-probe methods.

3.
Phys Chem Chem Phys ; 17(2): 762-82, 2015 Jan 14.
Article in English | MEDLINE | ID: mdl-25412176

ABSTRACT

Although protein folding is often described by motion on a funnel-shaped overall topology of the energy landscape, the many local interactions that can occur result in considerable landscape roughness which slows folding by increasing internal friction. Recent experimental results have brought to light that this roughness also causes unusual diffusional behaviour of the backbone of an unfolded protein, i.e. the relative motion of protein sections cannot be described by the normal diffusion equation, but shows strongly subdiffusional behaviour with a nonlinear time dependence of the mean square displacement, 〈r(2)(t)〉∝t(α) (α≪ 1). This results in significantly slower configurational equilibration than had been assumed hitherto. Analysis of the results also allows quantification of the energy landscape roughness, i.e. the root-mean-squared depth of local minima, yielding a value of 4-5kBT for a typical small protein.


Subject(s)
Peptides/chemistry , Proteins/chemistry , Amino Acid Sequence , Diffusion , Molecular Sequence Data , Movement , Peptides/metabolism , Protein Folding , Proteins/metabolism , Thermodynamics
4.
Nat Methods ; 11(11): 1131-4, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25282611

ABSTRACT

We describe a method for performing time-resolved X-ray crystallographic experiments based on the Hadamard transform, in which time resolution is defined by the underlying periodicity of the probe pulse sequence, and signal/noise is greatly improved over that for the fastest pump-probe experiments depending on a single pulse. This approach should be applicable on standard synchrotron beamlines and will enable high-resolution measurements of protein and small-molecule structural dynamics. It is also applicable to other time-resolved measurements where a probe can be encoded, such as pump-probe spectroscopy.


Subject(s)
Crystallography, X-Ray/methods , Plant Proteins/chemistry , Protein Conformation , Signal-To-Noise Ratio , Time Factors
5.
J Chem Phys ; 137(16): 165102, 2012 Oct 28.
Article in English | MEDLINE | ID: mdl-23126740

ABSTRACT

Molecular dynamics (MD) methods are increasingly widespread, but simulation of rare events in complex molecular systems remains a challenge. We recently introduced the boxed molecular dynamics (BXD) method, which accelerates rare events, and simultaneously provides both kinetic and thermodynamic information. We illustrate how the BXD method may be used to obtain high-resolution kinetic data from explicit MD simulations, spanning picoseconds to microseconds. The method is applied to investigate the loop formation dynamics and kinetics of cyclisation for a range of polypeptides, and recovers a power law dependence of the instantaneous rate coefficient over six orders of magnitude in time, in good agreement with experimental observations. Analysis of our BXD results shows that this power law behaviour arises when there is a broad and nearly uniform spectrum of reaction rate coefficients. For the systems investigated in this work, where the free energy surfaces have relatively small barriers, the kinetics is very sensitive to the initial conditions: strongly non-equilibrium conditions give rise to power law kinetics, while equilibrium initial conditions result in a rate coefficient with only a weak dependence on time. These results suggest that BXD may offer us a powerful and general algorithm for describing kinetics and thermodynamics in chemical and biochemical systems.


Subject(s)
Molecular Dynamics Simulation , Peptides/chemistry , Amino Acid Sequence , Cyclization , Kinetics , Molecular Sequence Data
6.
Proc Natl Acad Sci U S A ; 109(48): 19563-8, 2012 Nov 27.
Article in English | MEDLINE | ID: mdl-23150572

ABSTRACT

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k(B)T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k(B)T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.


Subject(s)
Protein Folding , Proteins/chemistry , Models, Molecular
7.
Biophys J ; 100(7): 1800-9, 2011 Apr 06.
Article in English | MEDLINE | ID: mdl-21463594

ABSTRACT

Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique-constant-deflection AFM-in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.


Subject(s)
Feedback , Lasers , Microscopy, Atomic Force/instrumentation , Protein Unfolding , Proteins/chemistry , Biomechanical Phenomena , Models, Molecular , Protein Refolding
8.
J Am Chem Soc ; 127(33): 11699-708, 2005 Aug 24.
Article in English | MEDLINE | ID: mdl-16104747

ABSTRACT

The conformation of a bisindolylmaleimide may be controlled by the size of a macrocyclic ring in which it is constrained. A range of techniques were used to demonstrate that the tether controls both the ratio of the two limiting conformers (syn and anti) in solution and the extent of conjugation between the maleimide and indole rings. Screening the conformationally diverse bisindolylmaleimides against a panel of protein kinases allowed their ATP binding sites to be compared using a chemical approach which, like sequence alignment, does not require detailed structural information. This approach lead to the conclusion that several AGC group protein kinases (including PKCalpha, PKCbeta, MSK1, p70 S6K, PDK-1, and MAPKAP-K1alpha) may be best inhibited by bisindolylmaleimides which adopt a compressed approximately C2-symmetric anti conformation; in constrast, GSK3beta may be best inhibited by bisindolylmaleimides whose ground state is a distorted syn conformation. It is concluded that PDK-1, whose structure has been determined by X-ray crystallography, and its mutants, may serve as particularly useful surrogates for the study of PKC inhibitors.


Subject(s)
Adenosine Triphosphate/chemistry , Indoles/chemistry , Maleimides/chemistry , Molecular Conformation , Protein Kinases/chemistry , Binding Sites , Indoles/chemical synthesis , Indoles/pharmacology , Ligands , Maleimides/chemical synthesis , Maleimides/pharmacology , Protein Kinases/drug effects , Protein Structure, Secondary , Stereoisomerism , Structure-Activity Relationship
9.
Biophys J ; 89(1): 506-19, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15863479

ABSTRACT

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.


Subject(s)
Bacterial Proteins/chemistry , Biophysics/methods , DNA-Binding Proteins/chemistry , Microscopy, Atomic Force/methods , Hydrogen Bonding , Kinetics , Models, Molecular , Models, Statistical , Monte Carlo Method , Peptostreptococcus/metabolism , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/chemistry , Stress, Mechanical , Temperature , Time Factors , Ubiquitin/chemistry
10.
Dalton Trans ; (2): 236-43, 2004 Jan 21.
Article in English | MEDLINE | ID: mdl-15356718

ABSTRACT

Crystals of the title compound (1) contain two independent, centrosymmetric half-molecules per asymmetric unit. While both of these show Jahn-Teller elongated six-coordinate geometries, the lengths of the elongated Cu-N bonds in the two molecules differ by 0.117(2) A at 30 K. The structure of one of these molecules (molecule A) does not vary with temperature below 350 K. The other molecule (molecule B) shows Cu-N bond lengths that are temperature-dependent between 225 and 375 K, but do not vary further at lower temperature. This indicates a fluxional axis of Jahn-Teller elongation in this molecule at these higher temperatures. Consideration of the thermal parameters in these structures implies that the fluxionality in molecule B is frozen out near 150 K. This conclusion is supported by a Q-band powder EPR study. The d-d transition energies of molecules A and B have been calculated by several density function (DF) methods, including a time-dependent DF calculation. The crystallographic data have been reproduced using the vibronic coupling model of Burgi and Hitchman. This has shown that the different fluxionality regimes for molecules A and B are not a consequence of their different static molecular structures, but rather reflect their different local environments in the crystal.

11.
J Mol Biol ; 342(1): 261-73, 2004 Sep 03.
Article in English | MEDLINE | ID: mdl-15313622

ABSTRACT

The four-helix protein Im7 folds through an on-pathway intermediate at pH 7.0 and 10 degrees C. By contrast, under these conditions there is no evidence for a populated intermediate in the folding of its more stable homologue, Im9, even in the presence of 0.4 M sodium sulphate. Previous studies using phi-value analysis have shown that the Im7 intermediate is misfolded, in that three of its four native helices are formed, but are docked in a non-native manner. Using knowledge of the structure of the intermediate of Im7, we have used rational design to stabilise an intermediate formed during the folding of Im9 by the introduction of specific stabilising interactions at positions known to stabilise the Im7 folding intermediate through non-native interactions. We show that the redesigned Im9 sequence folds with three-state kinetics at pH 7.0 and have used phi-value analysis to demonstrate that this species resembles the misfolded intermediate populated during Im7 folding. The redesigned Im9 sequence folds 20-fold faster than the wild-type protein under conditions in which folding is two-state. The data show that intermediate formation is an important feature of folding, even for small proteins such as Im9 for which these partially folded states do not become significantly populated. In addition, they show that the introduction of stabilising interactions can lead to rapid refolding, even when the contacts introduced are non-native.


Subject(s)
Bacterial Proteins , Colicins/chemistry , Protein Conformation , Protein Folding , Amino Acid Sequence , Colicins/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Denaturation , Sequence Alignment , Thermodynamics
12.
Chemistry ; 10(7): 1705-10, 2004 Apr 02.
Article in English | MEDLINE | ID: mdl-15054757

ABSTRACT

A new series of photocleavable protein cross-linking reagents based on bis(maleimide) derivatives of diaryl disulfides have been synthesised. They have been functionalised with cysteine and transient absorption spectra for the photolysis reaction have been recorded by using the pump-probe technique with a time resolution of 100 femtoseconds. Photolysis of the disulfide bond yields the corresponding thiyl radicals in less than a picosecond. There is a significant amount of geminate recombination, but some of the radicals escape the solvent cage and the quantum yield for photocleavage is 30 % in water.


Subject(s)
Cross-Linking Reagents/classification , Cross-Linking Reagents/chemical synthesis , Proteins/chemistry , Sulfhydryl Compounds/classification , Sulfhydryl Compounds/chemical synthesis , Cross-Linking Reagents/radiation effects , Free Radicals/chemical synthesis , Free Radicals/classification , Free Radicals/radiation effects , Models, Molecular , Molecular Structure , Oxidation-Reduction , Photochemistry , Sulfhydryl Compounds/radiation effects , Ultraviolet Rays
13.
J Mol Biol ; 337(1): 183-93, 2004 Mar 12.
Article in English | MEDLINE | ID: mdl-15001361

ABSTRACT

The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (deltadeltaG(ui) = 4.4 kJ/mol) but the native state is only slightly destabilised (deltadeltaG(nu) = 1.8 kJ/mol) at 10 degrees C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Phi-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Hydrogen/metabolism , Protein Structure, Secondary , Bacterial Proteins/genetics , Hydrogen/chemistry , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Mutation , Protein Denaturation , Protein Folding
14.
J Am Chem Soc ; 126(1): 336-43, 2004 Jan 14.
Article in English | MEDLINE | ID: mdl-14709100

ABSTRACT

Energy migration between tryptophan residues has been experimentally demonstrated in self-assembled peptide tapes. Each peptide contains 11 amino acids with a Trp at position 6. The peptide self-assembly is pH-sensitive and forms amphiphilic tapes, which further stack in ribbons (double tapes) and fibrils in water depending on the concentration. Fluorescence spectra, quenching, and anisotropy experiments showed that when the pH is lowered from 9 to 2, the peptide self-assembly buries the tryptophan in a hydrophobic and restricted environment in the interior of stable ribbons as expected on the basis of the peptide design. These fluorescence data support directly and for the first time the presence of such ribbons which are characterized by a highly packed and stable hydrophobic interior. In common with Trp in many proteins, fluorescence lifetimes are nonexponential, but the average lifetime is shorter at low pH, possibly due to quenching with neighboring Phe residues. Unexpectedly, time-resolved fluorescence anisotropy does not change significantly with self-assembly when in water. In highly viscous sucrose-water mixtures, the anisotropy decay at low pH was largely unchanged compared to that in water, whereas at high pH, the anisotropy decay increased significantly. We concluded that depolarization at low pH was not due to rotational diffusion but mainly due to energy migration between adjacent tryptophan residues. This was supported by a master equation kinetic model of Trp-Trp energy migration, which showed that the simulated and experimental results are in good agreement, although on average only three Trp residues were visited before emission.


Subject(s)
Oligopeptides/chemistry , Tryptophan/chemistry , Computer Simulation , Fluorescence Polarization , Hydrogen-Ion Concentration , Models, Molecular , Protein Structure, Secondary , Thermodynamics
15.
Anal Chem ; 75(16): 4182-7, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-14632133

ABSTRACT

In time-resolved, single-photon counting experiments, the standard method of nonlinear least-squares curve fitting incorrectly estimates the fluorescence lifetimes. Even for single-exponential data, errors may be up to +/- 15%, and for more complex fits, may be even higher, although the fitted line appears to describe the data. The origin of this error is not a result of the Poisson distribution, as is often stated, but is entirely due to the weighting of the fit. An alternative weighting method involving a minor change in the fitting method eliminates this problem, enabling accurate fitting even in difficult cases, including the small data sets observed in single molecule experiments and with a precision similar to that of maximum likelihood methods.

16.
Nat Struct Biol ; 10(9): 731-7, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12923573

ABSTRACT

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.


Subject(s)
Protein Structure, Secondary , Proteins/chemistry , Anisotropy , Escherichia coli/metabolism , Kinetics , Models, Molecular , Monte Carlo Method , Protein Folding , Protein Structure, Tertiary , Software , Temperature , Thermodynamics
17.
Philos Trans A Math Phys Eng Sci ; 361(1805): 713-28; discussion 728-30, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12871620

ABSTRACT

Understanding the mechanisms of protein folding is a major challenge that is being addressed effectively by collaboration between researchers in the physical and life sciences. Recently, it has become possible to mechanically unfold proteins by pulling on their two termini using local force probes such as the atomic force microscope. Here, we present data from experiments in which synthetic protein polymers designed to mimic naturally occurring polyproteins have been mechanically unfolded. For many years protein folding dynamics have been studied using chemical denaturation, and we therefore firstly discuss our mechanical unfolding data in the context of such experiments and show that the two unfolding mechanisms are not the same, at least for the proteins studied here. We also report unexpected observations that indicate a history effect in the observed unfolding forces of polymeric proteins and explain this in terms of the changing number of domains remaining to unfold and the increasing compliance of the lengthening unstructured polypeptide chain produced each time a domain unfolds.


Subject(s)
Protein Denaturation , Protein Folding , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Monte Carlo Method , Stress, Mechanical , Temperature
18.
Photochem Photobiol Sci ; 2(5): 585-90, 2003 May.
Article in English | MEDLINE | ID: mdl-12803082

ABSTRACT

The excited singlet state of an azomethine ylide or 'stable dipole' exhibits an ultrafast radiationless relaxation after femtosecond laser excitation. These transients are observed before the excited state decays in an almost activationless manner, the barrier is 440 cm-1, to the ground state with a 1.5 ps lifetime. Cooling of the hot ground state is also apparent in the transient absorption data and in methanol decays with a 5.7 ps lifetime. The viscosity dependence of the fluorescence yield and lifetime in different solvents is small and far less pronounced than in triphenylmethane dyes. Surprisingly, the excited state decay is not due to twisting about the C-N bond of the ylide but it is caused by buckling of one of the rings as the nitrogen atom changes character from sp2 to sp3 hybridisation.

19.
Protein Sci ; 11(12): 2759-65, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12441375

ABSTRACT

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.


Subject(s)
Protein Folding , Proteins/chemistry , Computer Simulation , Models, Molecular , Monte Carlo Method , Protein Structure, Tertiary , Proteins/metabolism
20.
Biophys J ; 83(1): 458-72, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12080133

ABSTRACT

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.


Subject(s)
Muscle Proteins/chemistry , Peptide Fragments/chemistry , Protein Kinases/chemistry , Proteins/chemistry , Amino Acid Sequence , Connectin , Dose-Response Relationship, Drug , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Monte Carlo Method , Muscle Proteins/metabolism , Mutation , Peptide Fragments/metabolism , Protein Denaturation , Protein Folding , Protein Kinases/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Thermodynamics
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