Darwin O. V. Alonso

The complete folding pathway of a protein from nanoseconds to microseconds

Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by φ values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native α-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion–collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution.

Mapping the early steps in the pH-induced conformational conversion of the prion protein

Under certain conditions, the prion protein (PrP) undergoes a conformational change from the normal cellular isoform, PrPC, to PrPSc, an infectious isoform capable of causing neurodegenerative diseases in many mammals. Conversion can be triggered by low pH, and in vivo this appears to take place in an endocytic pathway and/or caveolae-like domains. It has thus far been impossible to characterize the conformational change at high resolution by experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed 10-ns molecular dynamics simulations of PrPC in water at neutral and low pH. The core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet-like structure increases both by lengthening of the native β-sheet and by addition of a portion of the N terminus to widen the sheet by another two strands. The side chain of Met-129, a polymorphic codon in humans associated with variant Creutzfeldt–Jakob disease, pulls the N terminus into the sheet. Neutralization of Asp-178 at low pH removes interactions that inhibit conversion, which is consistent with the Asp-178–Asn mutation causing human prion diseases.

Ultrafast folding of α3D: A de novo designed three-helix bundle protein

Here, we describe the folding/unfolding kinetics of α3D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 ± 1.2 μs (at ≈50°C), indicating that a protein can fold on the 1- to 5-μs time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably ≥1 (μs)–1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. α3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.

PrPc glycoform heterogeneity as a function of brain region : Implications for selective targeting of neurons by prion strains

We recently found that deletion of the Asn-linked carbohydrate (CHO) at residue 197 of Syrian hamster (SHa) PrP(C) while retaining the CHO at Asn 181 has a profound effect on which population of neurons are targeted for conversion of SHaPrP(C) to SHaPrP(Sc) in transgenic (Tg) mice inoculated with scrapie prions. We hypothesized that selective targeting of neuronal populations is determined by cell-specific differences in the affinity of an infecting PrP(Sc) (prion) for PrP(C) and that the affinity might be modulated by nerve cell-specific differences in PrP(C) glycosylation. Here we tested this hypothesis by assessing whether or not each brain region in Syrian hamsters synthesizes different PrP(C) glycoforms, as inferred from 2D-gel electrophoresis. Reproducible differences in the number and isoelectric point of PrP(C) charge isomers were found as a function of brain region. The results of this study support the hypothesis that the PrP(Sc) accumulation and the vacuolation pattern phenotypes in the brain are governed by neuron-specific differences in PrP(C) glycoforms.

The hydration of amides in helices; a comprehensive picture from molecular dynamics, IR, and NMR

We examined the hydration of amides of α3D, a simple, designed three‐helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy (∼50%–70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I′ band) of a group of surface Ala residues. The surface‐exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I′ bands of the buried 13C‐labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen‐deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4–7 residues N‐terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 310/α/π‐helical conformation.

The intrinsic conformational propensities of the 20 naturally occurring amino acids and reflection of these propensities in proteins

Here, we compare the distributions of main chain (Φ,Ψ) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of Gly-Gly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X = the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (iii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the (Φ,Ψ) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation, and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the “intrinsic propensities” of amino acids for particular (Φ,Ψ) conformations. We also compare our helical propensities with experimental consensus values using the host–guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the “random coil” state.

The role of α‐, 310‐, and π‐helix in helix→coil transitions

The conformational equilibrium between 310‐ and α‐helical structure has been studied via high‐resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac‐AMAAKAWAAKA AAARA‐NH2. Their 750‐MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 310‐helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous αN(i,i + 2) and αN(i,i + 4) NOE cross‐peaks was proposed to represent conformational averaging between 310‐ and α‐helical structures. In this study, we describe 25‐nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 Å and a 10 Å force‐shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the 3JHNα coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i → i + 3 hydrogen bonds (310‐helix); however, there was a large population of bifurcated i → i + 3 and i → i + 4 α‐helical hydrogen bonds. In addition, both simulations contained considerable populations of π‐helix (i → i + 5 hydrogen bonds). Individual turns formed over residues 1–9, which we predict contribute to the intensities of the experimentally observed αN(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages.

Dynameomics: mass annotation of protein dynamics and unfolding in water by high-throughput atomistic molecular dynamics simulations

The goal of Dynameomics is to perform atomistic molecular dynamics (MD) simulations of representative proteins from all known folds in explicit water in their native state and along their thermal unfolding pathways. Here we present 188-fold representatives and their native state simulations and analyses. These 188 targets represent 67% of all the structures in the Protein Data Bank. The behavior of several specific targets is highlighted to illustrate general properties in the full dataset and to demonstrate the role of MD in understanding protein function and stability. As an example of what can be learned from mining the Dynameomics database, we identified a protein fold with heightened localized dynamics. In one member of this fold family, the motion affects the exposure of its phosphorylation site and acts as an entropy sink to offset another portion of the protein that is relatively immobile in order to present a consistent interface for protein docking. In another member of this family, a polymorphism in the highly mobile region leads to a host of disease phenotypes. We have constructed a web site to provide access to a novel hybrid relational/multidimensional database (described in the succeeding two papers) to view and interrogate simulations of the top 30 targets: http://www.dynameomics.org. The Dynameomics database, currently the largest collection of protein simulations and protein structures in the world, should also be useful for determining the rules governing protein folding and kinetic stability, which should aid in deciphering genomic information and for protein engineering and design.

Simulations of biomolecules: characterization of the early steps in the pH-induced conformational conversion of the hamster, bovine and human forms of the prion protein

As computer power increases, so too does the range of interesting biomolecular phenomena and properties that can be simulated. It is now possible to simulate complicated protein conformational changes at ambient or physiological temperatures. In this regard, we are attempting to map the conformational transitions of the normal, cellular prion protein (PrPC) to its infectious scrapie isoform (PrPSc), which causes neurodegenerative diseases in many mammals. These two forms have identical sequences and are conformational isomers, with heightened formation of β–sheet structure in the scrapie form. Conversion can be triggered by lowering the pH, but thus far it has been impossible to characterize the conformational change at high resolution using experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed molecular–dynamics simulations of hamster, human and bovine forms of the prion protein in water at neutral and low pH. In all cases the core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet–like structure increases both by lengthening of the native β–sheet and by addition of a portion of the N–terminus to widen the sheet by another 2–3 strands.

A microscopic view of peptide and protein solvation

The structure and dynamics of the water hydrating peptides and proteins are examined here at atomic resolution via molecular dynamics simulations. Detailed solvation density and residence time data for all 20 L-amino acids in an end-capped AXA tripeptide motif are presented. In addition, the solvation of the protein chymotrypsin inhibitor 2 is investigated as a point of comparison. Residues on the surface of proteins are not isolated; they interact both locally and non-locally in sequence space, and comparison of the solvation properties of each amino acid in both the peptide and protein allow us to distinguish inherent solvation properties from context-dependent perturbations due to neighboring residues. This work moves beyond traditional radial distribution functions and presents graphical representations of preferential solvation and orientation of water by side chains and the main chain. The combination of 0.3 micros of simulation data improves the statistical sampling over previous studies and reveals the significance of bridging water molecules that stabilize and mediate side chain-side chain, side chain-main chain and main chain-main chain interactions at the solvation interface.