Because conventional 1H NMR sequential assignment methods cannot readily be applied under physiol. These studies confirm and extend under physiol. Implications or the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogs. The soln. This spectroscopic comparison is of interest since the structure of the C-terminal region of the B-chain has not been detd. The present NMR studies are conducted in the presence of an org. The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their resp.
However, differences between insulin and DPI are obsd. Residues BB28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallog. This configuration differs from that described in a more org. The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition.
This paper describes the first detailed anal. Such an anal. Principles of Protein Structural Ensemble Determination. Bonomi, Massimiliano; Heller, Gabriella T. Elsevier Ltd. The biol. This aspect is particularly evident for disordered proteins, which constitute perhaps one-third of the human proteome.
Therefore, structural ensembles often offer more useful representations of proteins than individual conformations. Here, we describe how the well-established principles of protein structure detn. These principles concern primarily how to deal with conformationally heterogeneous states, and with exptl. We first review the growing literature of recent methods that combine exptl.
We then address some conceptual problems in the detn. Dynamic personalities of proteins. Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromols. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character or 'personality'.
The dream is to 'watch' proteins in action in real time at at. This requires the addn. This method continues to offer rich insights into the nature of IDPs in soln.
Substantial advances have been made in recent years in studies of proteins contg. These sequences are relevant to disease states such as Alzheimer's, Parkinson's, and Huntington's diseases, where disordered proteins misfold into harmful amyloid. Innovative applications of NMR are providing novel insights into mechanisms of protein aggregation and the complexity of IDP interactions with their targets.
As a basis for understanding the soln. A broad range of questions in the diversifying field of protein folding have been addressed with single-mol. Building on more than a decade of rapid method development, these techniques can now be used to investigate a wide span of timescales, an aspect that we focus on in this review. Important current topics range from the structure and dynamics of unfolded and intrinsically disordered proteins, including the coupling of folding and binding, to transition path times, the folding and misfolding of larger proteins, and their interactions with mol.
National Academy of Sciences. Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast nos.
A dye pair was specifically attached to the enzyme to measure structural changes through Foerster resonance energy transfer FRET. Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled the authors to monitor conformational changes of individual proteins for several hundred seconds.
FRET efficiency histograms were calcd. They showed an expansion of the unfolded proteins with increasing GdnHCl concn. Cross-correlation anal. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme mols. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chem.
Royal Society of Chemistry. Proper protein function in cells, tissues and organisms depends critically on correct protein folding or interaction with partners. Over the last decade, single-mol. FRET smFRET has emerged as a powerful tool to probe complex distributions, dynamics, pathways and landscapes in protein folding and binding reactions, leveraging its ability to avoid averaging over an ensemble of mols.
While smFRET was practiced in a two-color form until recently, the last few years have seen the development of enhanced multicolor smFRET methods that provide addnl. In this review, we provide a brief introduction to the smFRET technique, then follow with advanced multicolor measurements and end with ongoing methodol.
Elsevier B. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting x-ray structure; the RMS fluctuations calcd. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms.
For the isolated monomer in soln. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the x-ray conformation after 0. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer or the dimer in our study , the C-terminal end of the monomer residues BB30 is rearranged to allow binding to the insulin receptor.
The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.
B , , — , DOI: The insulin monomer contains two distinct surfaces, namely, the dimer forming surface DFS and the hexamer forming surface HFS , that are specifically designed to facilitate the formation of the dimer and the hexamer, resp.
In order to characterize the structural and dynamical behavior of interfacial water mols. Dynamical characterization reveals that the structural relaxation of the hydrogen bonds formed between the residues of DFS and the interfacial water mols. Furthermore, the residence times of water mols.
In particular, we find that more structured water mols. A significant slowing down is obsd. The surface topog. HFS having a large polar solvent accessible surface area and a convex extensive nonpolar region, drives the surrounding water mols.
In contrast, near the DFS, the surrounding water mols. We have followed escape trajectory of several such quasi-bound water mols. Cell Press. Earliest events in the aggregation process, such as single mol.
To this end, we have used well-tempered bias exchange metadynamics simulations to det. A bin-based clustering method that uses statistics accumulated in bias exchange metadynamics trajectories was employed to construct a detailed thermodn. The highest lifetime, lowest free-energy ensemble identified consisted of native conformations adopted by a folded insulin monomer in soln.
The lowest free-energy structure had a root mean square deviation of only 0. The second longest-lived metastable state was an unfolded, compact monomer with little similarity to the native structure. We have identified three addnl. We then carried out an exhaustive structural characterization of metastable states on the basis of tertiary contact maps and per-residue accessible surface areas. We have also detd. The ensemble contg. We have discussed the relative importance of nativelike vs.
We also provide a simple measure to det. Finally, we have compared and contrasted this intermediate with exptl. Elsevier Science B. The effectiveness of a new algorithm, parallel tempering, is studied for numerical simulations of biol. These mols. The resulting slowing down in numerical simulations is overcome by the new method. This is demonstrated by performing simulations with high statistics for one of the simplest peptides, Met-enkephalin.
The numerical effectiveness of the new technique was found to be much better than traditional methods and is comparable to sophisticated methods like generalized ensemble techniques. We discuss the history of the parallel tempering simulation method. From its origins in data anal. We discuss the theory behind the method and its various generalizations. We mention a selected set of the many applications that have become possible with the introduction of parallel tempering, and we suggest several promising avenues for future research.
American Institute of Physics. A review; Markov state models of mol. This approach has many appealing characteristics compared to straightforward mol. In this paper, we summarize the current state of the art in generation and validation of MSMs and give some important new results. We describe an upper bound for the approxn. In contrast to previous practice, it becomes clear that the best MSM is not obtained by the most metastable discretization, but the MSM can be much improved if non-metastable states are introduced near the transition states.
Moreover, we show that it is not necessary to resolve all slow processes by the state space partitioning, but individual dynamical processes of interest can be resolved sep. We also present an efficient estimator for reversible transition matrixes and a robust test to validate that a MSM reproduces the kinetics of the mol.
Variational Approach to Molecular Kinetics. Theory Comput. Nueske, Feliks; Keller, Bettina G. The eigenvalues and eigenvectors of the mol. This includes the stationary distribution, the metastable states, and state-to-state transition rates.
Here, we present a variational approach for computing these dominant eigenvalues and eigenvectors. This approach is analogous to the variational approach used for computing stationary states in quantum mechanics. A corresponding method of linear variation is formulated. It is shown that the matrixes needed for the linear variation method are correlation matrixes that can be estd. The method proposed here is thus to first define a basis set able to capture the relevant conformational transitions, then compute the resp.
One approach to analyzing the dynamics of a phys. This approach has been particularly successful for mol. Detecting such patterns is the central objective of the variational approach to conformational dynamics VAC , as well as the related methods of time-lagged independent component anal. In VAC, the search for slowly decorrelating patterns is formalized as a variational problem solved by the eigenfunctions of the system's transition operator.
VAC computes solns. Here, we build on VAC's success by addressing two practical limitations. First, VAC can give poor eigenfunction ests.
Second, VAC can overfit when using flexible parametrizations such as artificial neural networks with insufficient regularization. IVAC integrates over multiple lag times before solving the variational problem, making its results more robust and reproducible than VAC's. Academic Press. Recently, a novel concept for the computation of essential features of the dynamics of Hamiltonian systems such as mol. The realization of this concept had been based on subdivision techniques applied to the Frobenius-Perron operator for the dynamical system.
The present paper suggests an alternative but related concept that merges the conceptual advantages of the dynamical systems approach with the appropriate statistical physics framework.
This approach allows us to define the phrase "conformation" in terms of the dynamical behavior of the mol. In a first step, the frequency of conformational changes is characterized in statistical terms leading to the definition of some Markov operator T that describes the corresponding transition probabilities within the canonical ensemble. In a second step, a discretization of T via specific hybrid Monte Carlo techniques is shown to lead to a stochastic matrix P.
With these theor. It is demonstrated that the discretization of T can be restricted to few essential degrees of freedom so that the combinatorial explosion of discretization boxes is prevented and biomol. Numerical results for the n-pentane mol.
MacKerell, A. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal bonding and interaction nonbonding terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions.
Optimization of the internal parameters used exptl. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the at. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in a crystal.
A detailed anal. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in soln. Extensive comparisons between mol. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of mols.
Best, Robert B. To overcome this, as well as make other improvements in the model, we present a combination of refinements that should result in enhanced accuracy in simulations of proteins. Side-chain torsion parameters have been optimized by fitting to backbone-dependent quantum-mech. A comprehensive validation of the revised force field was then performed against a collection of exptl.
The results indicate that the revised CHARMM 36 parameters represent an improved model for modeling and simulation studies of proteins, including studies of protein folding, assembly, and functionally relevant conformational changes.
Methods , 14 , 71 — 73 , DOI: SoftwareX , , 19 — 25 , DOI: The web environment provides an ideal platform to build and validate a mol. Proper treatment of nonbonded interactions is essential for the accuracy of mol. A wide range of Lennard-Jones LJ cutoff schemes and integrator algorithms were tested to find the optimal simulation protocol to best match bilayer properties of six lipids with varying acyl chain satn. MD simulations of a 1,2-dipalmitoyl-sn-phosphatidylcholine DPPC bilayer were used to obtain the optimal protocol for each program.
MD simulations with all programs were found to reasonably match the DPPC bilayer properties surface area per lipid, chain order parameters, and area compressibility modulus obtained using the std. The optimal simulation protocol was then applied to the other five lipid simulations and resulted in excellent agreement between results from most simulation programs as well as with exptl.
AMBER compared least favorably with the expected membrane properties, which appears to be due to its use of the hard-truncation in the LJ potential vs. This protocol is expected to be applicable to the remainder of the additive C36 FF including the proteins, nucleic acids, carbohydrates, and small mols.
Graphics , 14 , 33 — 38 , DOI: VMD is a mol. VMD can simultaneously display any no. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback.
VMD has also been expressly designed with the ability to animate mol. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biol. Goga, N. In this article, we present several algorithms for stochastic dynamics, including Langevin dynamics and different variants of Dissipative Particle Dynamics DPD , applicable to systems with or without constraints.
The algorithms are based on the impulsive application of friction and noise, thus avoiding the computational complexity of algorithms that apply continuous friction and noise. We show that the measured thermal relaxation rates agree well with theor. The influence of various parameters on the diffusion coeff. The algorithm is inherently stable, as the constraints themselves are reset instead of derivs.
Although the derivation of the algorithm is presented in terms of matrixes, no matrix matrix multiplications are needed and only the nonzero matrix elements have to be stored, making the method useful for very large mols.
Parallelization of the algorithm is straightforward. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolution using fast Fourier transforms. Timings and accuracies are presented for three large cryst.
Jorgensen, William L. Classical Monte Carlo simulations were carried out for liq. Comparisons were made with exptl. The computed densities and potential energies agree with expt. The discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations.
Comparisons were made for the self-diffusion coeffs. Sondergaard, Chresten R. The authors present new algorithms that allow pKa shifts due to inductive i. The no. Finally, the authors present a novel algorithm that identifies noncovalently coupled ionizable groups, where pKa prediction may be esp.
Olsson, Mats H. The authors have revised the rules and parameters for one of the most commonly used empirical pKa predictors, PROPKA, based on better phys. The authors have introduced a new and consistent approach to interpolate the description between the previously distinct classifications into internal and surface residues, which otherwise is found to give rise to an erratic and discontinuous behavior.
Since the goal of this study is to lay out the framework and validate the concept, it focuses on Asp and Glu residues where the protein pKa values and structures are assumed to be more reliable. The new and improved implementation is evaluated and discussed; it is found to agree better with expt.
The most significant advance, however, is in reducing the no. A Lagrangian formulation is introduced; it can be used to make mol. In this formulation the MD cell shape and size can change according to dynamic equations given by this Lagrangian. This MD technique was used to the study of structural transitions of a Ni single crystal under uniform uniaxial compressive and tensile loads. Some results regarding the stress-strain relation obtained by static calcns.
Under compressive loading, the model of Ni shows a bifurcation in its stress-strain relation; this bifurcation provides a link in configuration space between cubic and hexagonal close packing. Such a transition could perhaps be obsd. A temperature predictor for parallel tempering simulations.
An algorithm is proposed that generates a set of temps. The input consists of the no. The temps. To facilitate its use, the algorithm has been implemented as a web server at. Scherer, Martin K.
Markov state models MSMs and related models of mol. However, the estn. PyEMMA can read all common mol. PCA and time-lagged independent component anal. Systematic model validation and error calcn. PyEMMA offers a wealth of anal. We have derived a systematic and accurate way to coarse-grain MSMs to few states and to illustrate the structures of the metastable states of the system.
Plotting functions to produce a manuscript-ready presentation of the results are available. In this work, we demonstrate the features of the software and show new methodol.
Hydrogen exchange HX studies have provided crit. The interpretation of the wealth of data generated by HX-MS expts. Most reported computational HX modeling studies have employed solvent-accessible-surface-area based metrics in attempts to interpret HX data on the basis of structures or models. A computational HX-MS prediction method based on classification of the amide hydrogen bonding modes mimicking the local unfolding model is demonstrated.
Predicted PFs are then used for calcg. High correlation between prediction and expt. Successful rate class decompns. This assessment is further strengthened in a comparison of residue resolved protection factor predictions for staphylococcal nuclease with NMR data, which was also used to compare prediction performance with other algorithms described in the literature.
The demonstrated transferable and scalable MD based HX prediction approach adds significantly to the available tools for HX-MS data interpretation based on available structures and models. American Society for Biochemistry and Molecular Biology. How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding.
Here, we employ chiral mutagenesis, comparison of corresponding D and L amino acid substitutions, to investigate a possible switch in the B-chain. Although the isomeric structures each retain a native-like overall fold, the L-SerB8 analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the D-SerB8 analog or native monomer.
Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. Elsevier Science Ltd. A review and discussion. How fast can a protein possibly fold. This question has stimulated researchers to seek fast-folding proteins and to engineer them to fold even faster. Proteins that fold at or near the speed limit are prime candidates for all-atom mol. They may also have no free energy barrier, allowing the direct observation of intermediate structures on the pathways from the unfolded to the folded state.
Both exptl. BBA - Biochim. Acta , 14 , — , DOI: A detn. Purified, dried pork I was treated with On the basis of a min. Assuming that the amide H atoms of the 48 peptide bonds are protected and considering that the peptide-bond H in shorter peptides like leucyltriglycine exchange readily though at a finite rate under the same conditions, the results confirm the existence of stable internal H bonds between the CO.
Study of the exchange of H atoms of the Na salt of isolated oxidized A-chain showed that all of the H atoms of the peptide bonds exchange in this substance. Hydrogen Exchange in Proteins. Protein Sci. Cambridge University Press. A review with 88 refs. This discussion, prepd. 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 equil.
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 biol.
Mohammadiarani, Hossein; Shaw, Vincent S. Hydrogen-deuterium exchange HDX expts. To predict the propensity of amide hydrogens for exchange with deuterium, several models have been reported in which computations of amide-hydrogen protection factors are carried out using mol. Given significant variation in the criteria used in different models, the robustness and broader applicability of these models to other proteins, esp.
The sensitivity of the predictions when MD simulations are conducted with different force-fields is yet to tested and quantified. Using MD simulations and exptl. HDX data on three homologous signaling proteins, we report detailed studies quantifying the performance of seven previously reported models M1-M7 of two general types: empirical and fractional-population models.
We find that the empirical models show inconsistent predictions but predictions of the fractional population models are robust. Contrary to previously reported work, we find that the solvent-accessible surface area of amide hydrogens is a useful metric when combined with a new metric defining the distances of amide hydrogens from the first polar atoms in proteins. On the basis of this, we report two new models, one empirical M8 and one population-based M9. We find strong protection of amide hydrogens from solvent exchange both within the stable helical motifs and also in the interhelical loops.
We further observe that the exchange-competent states of amide hydrogens occur on the sub ps time-scale via localized fluctuations, and such states among amides of a given protein do not appear to show any cooperativity or allosteric coupling.
Acid Stabilization of Insulin. Biochemistry , 32 , — , DOI: Bryant, Christopher; Spencer, Donald B. The effect of pH on the conformational stability of insulin was studied. PH titrn. The acid stabilization of insulin's conformation was attributed to the protonation of histidine at position 5 on the B-chain HB5 as detd. Further acidification at least to pH 2 does not decrease the free energy of unfolding.
A conformational change in the tertiary structure, as indicated by the near-UV CD spectrum, accompanies this change in stability. The authors propose that this acid stabilization of insulin is physiol.
Intrinsically disordered proteins IDPs are notoriously challenging to study both exptl. The structure of IDPs cannot be described by a single conformation but must instead be described as an ensemble of interconverting conformations. Atomistic simulations are increasingly used to obtain such IDP conformational ensembles.
Ensembles obtained with different force fields exhibited marked differences in chain dimensions, H-bonding, and secondary structure content. These differences were unexpectedly large: changing the force field was found to have a stronger effect on secondary structure content than changing the entire peptide sequence. To eliminate inadequate sampling as a reason for differences between force fields, extensive simulations were carried out 0.
The findings highlighted how IDPs, with their rugged energy landscapes, are highly sensitive test systems that are capable of revealing force field deficiencies and, therefore, contributing to force field development.
Biophysical Characterization of Intrinsically Disordered Proteins. The challenges assocd. NMR spectroscopy is perhaps the most readily suited technique for providing high-resoln. Optical methods, solid state NMR, ESR and X-ray scattering can also provide valuable information regarding the ensemble of conformations sampled by disordered states.
Finally, computational studies have begun to assume an increasingly important role in interpreting and extending the impact of exptl. This article discusses recent advances in the applications of these methods to intrinsically disordered proteins. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chem. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein mol.
This work illustrates the utility of chem. Smith, Adam W. Data were interpreted on the basis of structure-based spectroscopic modeling of conformers obtained from extensive mol. The data indicate that structures at low temp.
As the temp. Hydrogen bonding contacts in the midstrand region remain at all temps. Our data show no evidence of an extended chain or random coil state for the TZ2 peptide at any temp. The methods demonstrated here offer an approach to characterizing conformational variation within the folded or unfolded states of proteins and peptides. Annual Reviews. Two-dimensional IR spectroscopy of amide I vibrations is increasingly being used to study the structure and dynamics of proteins and peptides.
Amide I, a primarily carbonyl stretching vibration of the protein backbone, provides information on secondary structures as a result of vibrational couplings and on hydrogen-bonding contacts when isotope labeling is used to isolate specific sites.
In parallel with expts. Mixed quantum-classical models use spectroscopic maps to translate the structural information into a quantum-mech. Hamiltonian for the spectroscopically obsd. This allows one to model the spectroscopy of large proteins, disordered states, and protein conformational dynamics.
With improvements in amide I models, quant. We review the advances in developing these models, their theor. In a recently reported study [Mukherjee, et al. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amt. In this study, we use mol.
We use two models to convert the simulations to frequency trajectories from which the mean frequencies, std. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model.
We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helixes, shifting the IR frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helixes are most ordered.
Comparison of the simulations to expt. Taken together, the simulations and expts. Reppert, Mike; Roy, Anish R. The characterization of intrinsically disordered protein IDP ensembles is complicated both by inherent heterogeneity and by the fact that many common exptl. For this reason, the development of alternative structural tools for probing IDP ensembles has attracted considerable attention.
Here the authors describe the recent work in developing exptl. In this approach, the IR absorption frequencies of isotope-labeled amide bonds probe their local electrostatic environments and structures. Empirical frequency maps allow us to use this spectroscopic data as a direct exptl.
The authors apply these methods to a family of short elastin-like peptides ELPs , fragments of the elastin protein based around the Pro-Gly turn motif characteristic of the elastomeric segments of the full protein. Using a max. The monomer-dimer transition of insulin has been probed with two-dimensional IR spectroscopy and related IR spectroscopies to isolate spectral signatures of the conformational changes concomitant with dissocn.
These expts. These spectral changes provided a structurally sensitive probe of dimer dissocn. The solvent conditions surveyed the effects of ethanol and salt addn.
It was found that addn. Simulation of the monomer 2D IR spectra indicates that the B-chain C terminus is partially disordered, although not fully solvated by water. The IR spectroscopy and dynamics of - CO labels in wild type and mutant insulin monomer and dimer are characterized from mol. It is found that the spectroscopy of monomeric and dimeric forms in the region of the amide-I vibration differs for residues BB26 and DD26, which are involved in dimerization of the hormone.
Also, the spectroscopic signatures change for mutations at position B24 from phenylalanine - which is conserved in many organisms and known to play a central role in insulin aggregation - to alanine or glycine. Using three different methods to det. The spectroscopic response of monomeric WT and mutant insulin differs from that of their resp. Although the crystal structure of the dimer is that of a sym.
Together with earlier work on the thermodn. IR spectroscopy provides a potentially powerful way to characterize the aggregation state and dimerization energy of modified insulins. The protein hormone insulin exists in various oligomeric forms, and a key step in binding its cellular receptor is dissocn. This dissocn. Despite its fundamental and practical importance, the mechanism of insulin dimer dissocn.
Here, we use mol. We find that the dissocn. Along one limiting path, the dissocn. We simulate IR spectroscopy expts.
The authors have studied the structure and conformational dynamics of insulin dimer using a Markov state model MSM built from extensive unbiased atomistic mol. The authors' model reveals two significant conformations to the dimer: a dominant native state consistent with other exptl. The twisted state primarily influences the contacts involving the C-terminus of insulin's B chain, shifting the registry of its intermol.
Computational amide I spectroscopy of site-specifically 13C18O labeled amides indicates that the native and twisted conformation can be distinguished through a series of single and dual labels involving the B24F, B25F, and B26Y residues. This study will provide important interpretive tools for IR spectroscopic studies of insulin structure and transient IR kinetics expts.
Menting, John G. Insulin receptor signaling has a central role in mammalian biol. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signaling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor IGF1R. Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein.
Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs.
Contact between insulin and L1 is restricted to insulin B-chain residues. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases.
We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titrn. Together, our findings provide an explanation for a wealth of biochem. Human type 1 insulin-like growth factor receptor is a homodimeric receptor tyrosine kinase that signals into pathways directing normal cellular growth, differentiation and proliferation, with aberrant signalling implicated in cancer.
Insulin-like growth factor binding is understood to relax conformational restraints within the homodimer, initiating transphosphorylation of the tyrosine kinase domains. However, no three-dimensional structures exist for the receptor ectodomain to inform atomic-level understanding of these events.
Here, we present crystal structures of the ectodomain in apo form and in complex with insulin-like growth factor I, the latter obtained by crystal soaking. These structures not only provide a wealth of detail of the growth factor interaction with the receptor's primary ligand-binding site but also indicate that ligand binding separates receptor domains by a mechanism of induced fit. Our findings are of importance to the design of agents targeting IGF-1R and its partner protein, the human insulin receptor.
Single Chain Des- B30 Insulin. Peptide Protein Res. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation.
Single-chain des- B30 porcine insulin SCI [] was synthesized from porcine insulin [] by trypsin [] in a medium with a low content of H2O. Trypsin catalyzed an intramol. The insulin deriv. The structure was proven by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degrdn.
The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form.
SCI had little effect on blood sugar in mice; the estd. A completely inactive analog. Derewenda, U. A crystal structure of a totally inactive insulin mol. For this insulin mol.
The mol. The hexamers of the crosslinked insulin mol. The structure was crystallog. Comparison of the BA1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The anal. This partial unfolding of the B-chain exposes an alternative protein surface for receptor binding and may explain its relatively high biol. M [ Crossref ], [ PubMed ], [ CAS ], Google Scholar 78 Enhancing the activity of a protein by stereospecific unfolding: conformational life cycle of insulin and its evolutionary origins.
A central tenet of mol. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biol. A model is provided by insulin, well characterized at. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element.
This strand is anchored by an invariant side chain PheB24 ; its substitution by Ala leads to an unstable but native-like analog of low activity.
Corresponding photoactivable derivs. L- or D-para-azido-Phe crosslink to the insulin receptor with higher D-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is assocd. Conservation of PheB24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface.
Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary mol. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences. Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved.
Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant PheB24, one of the key residues of the hormone, in this process remains unclear.
For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, the authors prepd. The inactive [HisB24]-insulin mol. The authors' data indicate the importance of the arom. Moreover, they also suggest limited, BB30 only, unfolding of the C terminus of the B-chain upon insulin activation.
It was found that PT insulin has a highly flexible structure in pure water and is present in at least two different conformations, although with an overall tertiary structure similar to that of native insulin. Furthermore, the native helical structures are poorly defined. Surprisingly, the mutant has a biol.
Thus, as shown by a detailed detn. Despite the key role of the insulin monomer in aggregation and fibril formation at interfaces, its presence is difficult to ascertain spectroscopically.
Ganim et al. For bulk solutions, a detailed analysis of circular dichroism CD spectra can also be used to derive the oligomeric composition, 10 but this method is not sensitive enough for interfaces. SFG has a sensitivity comparable to infrared spectroscopy, 15 but possesses the added advantage that it is surface specific due to its optical selection rules.
As a second order nonlinear optical process, where an infrared and a visible photon are absorbed and an anti-Stokes Raman photon is emitted, SFG requires a lack of inversion symmetry to generate a signal.
It therefore only detects vibrations from molecular layers with a net orientation at interfaces. Briefly, the SFG process consists of mixing two laser pulses at an interface see Fig. A broadband tunable IR pulse and a narrowband visible pulse are overlapped temporally and spatially onto a surface.
Photons at the sum frequency wavelength are generated by oriented interfacial molecules only and detected. We chose to adsorb from solution to better represent conditions found during agitation-accelerated insulin fibrillation.
The spectra have been measured using ssp polarisation combination as sketched in Fig. In Fig. The amide I band lies in both cases at the same frequency, which is characteristic for the mostly helical structure of HI in its native state.
The difference to the bulk FT-IR spectrum is caused by the requirement of the detected vibration to be both infrared and Raman active, which changes the overall appearance of the amide I mode.
The integrated peak intensities are very similar, specifically 8. This suggests that only one species of insulin saturates the interface already at low bulk concentration and this species is most likely the monomer. How can we tell which oligomeric species is bound to the surface?
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