Patrick J. Fleming

A backbone-based theory of protein folding.

Under physiological conditions, a protein undergoes a spontaneous disorder ⇌ order transition called “folding.” The protein polymer is highly flexible when unfolded but adopts its unique native, three-dimensional structure when folded. Current experimental knowledge comes primarily from thermodynamic measurements in solution or the structures of individual molecules, elucidated by either x-ray crystallography or NMR spectroscopy. From the former, we know the enthalpy, entropy, and free energy differences between the folded and unfolded forms of hundreds of proteins under a variety of solvent/cosolvent conditions. From the latter, we know the structures of ≈35,000 proteins, which are built on scaffolds of hydrogen-bonded structural elements, α-helix and β-sheet. Anfinsen showed that the amino acid sequence alone is sufficient to determine a proteins structure, but the molecular mechanism responsible for self-assembly remains an open question, probably the most fundamental open question in biochemistry. This perspective is a hybrid: partly review, partly proposal. First, we summarize key ideas regarding protein folding developed over the past half-century and culminating in the current mindset. In this view, the energetics of side-chain interactions dominate the folding process, driving the chain to self-organize under folding conditions. Next, having taken stock, we propose an alternative model that inverts the prevailing side-chain/backbone paradigm. Here, the energetics of backbone hydrogen bonds dominate the folding process, with preorganization in the unfolded state. Then, under folding conditions, the resultant fold is selected from a limited repertoire of structural possibilities, each corresponding to a distinct hydrogen-bonded arrangement of α-helices and/or strands of β-sheet.

Polyproline II helix is the preferred conformation for unfolded polyalanine in water

Does aqueous solvent discriminate among peptide conformers? To address this question, we computed the solvation free energy of a blocked, 12‐residue polyalanyl‐peptide in explicit water and analyzed its solvent structure. The peptide was modeled in each of 4 conformers: α‐helix, antiparallel β‐strand, parallel β‐strand, and polyproline II helix (PII). Monte Carlo simulations in the canonical ensemble were performed at 300 K using the CHARMM 22 forcefield with TIP3P water. The simulations indicate that the solvation free energy of PII is favored over that of other conformers for reasons that defy conventional explanation. Specifically, in these 4 conformers, an almost perfect correlation is found between a residues solvent‐accessible surface area and the volume of its first solvent shell, but neither quantity is correlated with the observed differences in solvation free energy. Instead, solvation free energy tracks with the interaction energy between the peptide and its first‐shell water. An additional, previously unrecognized contribution involves the conformation‐dependent perturbation of first‐shell solvent organization. Unlike PII, β‐strands induce formation of entropically disfavored peptide:water bridges that order vicinal water in a manner reminiscent of the hydrophobic effect. The use of explicit water allows us to capture and characterize these dynamic water bridges that form and dissolve during our simulations. Proteins 2004.

Do all backbone polar groups in proteins form hydrogen bonds

Evidence from proteins and peptides supports the conclusion that intrapeptide hydrogen bonds stabilize the folded form of proteins. Paradoxically, evidence from small molecules supports the opposite conclusion, that intrapeptide hydrogen bonds are less favorable than peptide–water hydrogen bonds. A related issue—often lost in this debate about comparing peptide–peptide to peptide– water hydrogen bonds—involves the energetic cost of an unsatisfied hydrogen bond. Here, experiment and theory agree that breaking a hydrogen bond costs between 5 and 6 kcal/mol. Accordingly, the likelihood of finding an unsatisfied hydrogen bond in a protein is insignificant. This realization establishes a powerful rule for evaluating protein conformations.

[8] Purification of cytochrome b5

Publisher Summary This chapter provides information on the purification of cytochrome b 5 . The isolation of cytochrome b 5 from liver endoplasmic reticulum in its complete form requires fractionation procedures with detergents at 0° to avoid loss of peptide fragments by endopeptidase activity. The chapter mentions the procedure for the heme peptide and NADH-cytochrome b 5 reductase that can also be used for the complete form of cytochrome b 5 . NADH and the reductase are added to the cytochrome to yield the reduced spectrum. This procedure is the one that gradually evolved from the original procedure described for rabbit liver. To scale the preparation to large amounts of yearling steer liver, CaCI 2 precipitation of microsomes and slightly different column chromatography steps are employed. Cytochrome b 5 serves as an electron acceptor for cytochrome b 5 reductase and an electron donor for stearyl-CoA desaturase where the binding of the protein to phospholipid.

The Protein Coil Library: A structural database of nonhelix, nonstrand fragments derived from the PDB

Approximately half the structure of folded proteins is either α‐helix or β‐strand. We have developed a convenient repository of all remaining structure after these two regular secondary structure elements are removed. The Protein Coil Library (http://roselab.jhu.edu/coil/) allows rapid and comprehensive access to non‐α‐helix and non‐β‐strand fragments contained in the Protein Data Bank (PDB). The library contains both sequence and structure information together with calculated torsion angles for both the backbone and side chains. Several search options are implemented, including a query function that uses output from popular PDB‐culling servers directly. Additionally, several popular searches are stored and updated for immediate access. The library is a useful tool for exploring conformational propensities, turn motifs, and a recent model of the unfolded state. Proteins 2005.

Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm

Thermodynamic stabilities are pivotal for understanding structure–function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure–stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.

A novel method reveals that solvent water favors polyproline II over β‐strand conformation in peptides and unfolded proteins: conditional hydrophobic accessible surface area (CHASA)

In aqueous solution, the ensemble of conformations sampled by peptides and unfolded proteins is largely determined by their interaction with water. It has been a long‐standing goal to capture these solute‐water energetics accurately and efficiently in calculations. Historically, accessible surface area (ASA) has been used to estimate these energies, but this method breaks down when applied to amphipathic peptides and proteins. Here we introduce a novel method in which hydrophobic ASA is determined after first positioning water oxygens in hydrogen‐bonded orientations proximate to all accessible peptide/protein backbone N and O atoms. This conditional hydrophobic accessible surface area is termed CHASA. The CHASA method was validated by predicting the polyproline‐II (PII) and β‐strand conformational preferences of non‐proline residues in the coil library (i.e., non‐α‐helix, non‐β‐strand, non‐β‐turn library derived from X‐ray elucidated structures). Further, the method successfully rationalizes the previously unexplained solvation energies in polyalanyl peptides and compares favorably with published experimentally determined PII residue propensities.

Secondary structure determines protein topology.

Using a test set of 13 small, compact proteins, we demonstrate that a remarkably simple protocol can capture native topology from secondary structure information alone, in the absence of long‐range interactions. It has been a long‐standing open question whether such information is sufficient to determine a proteins fold. Indeed, even the far simpler problem of reconstructing the three‐dimensional structure of a protein from its exact backbone torsion angles has remained a difficult challenge owing to the small, but cumulative, deviations from ideality in backbone planarity, which, if ignored, cause large errors in structure. As a familiar example, a small change in an elbow angle causes a large displacement at the end of your arm; the longer the arm, the larger the displacement. Here, correct secondary structure assignments (α‐helix, β‐strand, β‐turn, polyproline II, coil) were used to constrain polypeptide backbone chains devoid of side chains, and the most stable folded conformations were determined, using Monte Carlo simulation. Just three terms were used to assess stability: molecular compaction, steric exclusion, and hydrogen bonding. For nine of the 13 proteins, this protocol restricts the main chain to a surprisingly small number of energetically favorable topologies, with the native one prominent among them.

Outer membrane phospholipase A in phospholipid bilayers: A model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers☆

Understanding the forces that stabilize membrane proteins in their native states is one of the contemporary challenges of biophysics. To date, estimates of side chain partitioning free energies from water to the lipid environment show disparate values between experimental and computational measures. Resolving the disparities is particularly important for understanding the energetic contributions of polar and charged side chains to membrane protein function because of the roles these residue types play in many cellular functions. In general, computational free energy estimates of charged side chain partitioning into bilayers are much larger than experimental measurements. However, the lack of a protein-based experimental system that uses bilayers against which to vet these computational predictions has traditionally been a significant drawback. Moon & Fleming recently published a novel hydrophobicity scale that was derived experimentally by using a host-guest strategy to measure the side chain energetic perturbation due to mutation in the context of a native membrane protein inserted into a phospholipid bilayer. These values are still approximately an order of magnitude smaller than computational estimates derived from molecular dynamics calculations from several independent groups. Here we address this discrepancy by showing that the free energy differences between experiment and computation become much smaller if the appropriate comparisons are drawn, which suggests that the two fields may in fact be converging. In addition, we present an initial computational characterization of the Moon & Fleming experimental system used for the hydrophobicity scale: OmpLA in DLPC bilayers. The hydrophobicity scale used OmpLA position 210 as the guest site, and our preliminary results demonstrate that this position is buried in the center of the DLPC membrane, validating its usage in the experimental studies. We further showed that the introduction of charged Arg at position 210 is well tolerated in OmpLA and that the DLPC bilayers accommodate this perturbation by creating a water dimple that allows the Arg side chain to remain hydrated. Lipid head groups visit the dimple and can hydrogen bond with Arg, but these interactions are transient. Overall, our study demonstrates the unique advantages of this molecular system because it can be interrogated by both computational and experimental practitioners, and it sets the stage for free energy calculations in a system for which there is unambiguous experimental data. This article is part of a Special Issue entitled: Membrane protein structure and function.

Ab Initio Protein Folding Using LINUS

Publisher Summary This chapter examines the various aspects of ab initio protein folding using LINUS. LINUS is an ab initio method for simulating the folding of a protein on the basis of simple physical principles. The approach emphasizes the organizing role of steric exclusion and conformational entropy in guiding folding. In addition to steric interactions, which are repulsive, LINUS also includes attractive forces resulting from hydrogen bonding and hydrophobic burial. A typical LINUS simulation begins with a sequence of interest modeled as an extended polypeptide chain with all nonhydrogen atoms included. The LINUS scoring functions components include hydrogen bonding, hydrophobic contacts, and backbone torsion. The villin headpiece subdomain is a small 36-residue protein containing three short helices and a hydrophobic core. The first and second helices are joined by a six-residue loop, and the second and third helices are joined by a three-residue segment. It is found that hydrophobic collapse to the overall villin headpiece structure is seen occasionally in the ensemble of saved conformations, but it does not persist for many cycles.