Side-chain Packing Research Articles

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.

Molecular Design and X-Ray Crystal Structure of Engineered Phospholamban Transmembrane Variant

Phospholamban is an oligomeric transmembrane (TM) protein thoroughly studied as a contractility regulator in cardiomyopathies and as a model system for spectroscopy and protein folding. While phospholamban forms a narrow apolar channel, it debated given conflicting biochemical studies and NMR-based models whether this protein simply regulates the SERCA2 Ca2+ pump or also conducts ions. Additionally, the “Leu-Ile zipper” motif at its core poses a puzzling paradigm in TM protein folding: how does hydrophobic side chain packing drive association of alpha-helices in the already hydrophobic lipid bilayer. Meanwhile, phospholamban's X-ray crystal structure has not been reported, but would provide invaluable answers. We re-designed this protein, guided by simulation, and solved the variant's structure. First, we conducted 300 ns all-atom molecular dynamics simulations in lipid bilayers to assess the structural rigidity and pore size of the phospholamban TM domain from a reported NMR-based model. We observed the channel to widen, but remain dehydrated. The N-terminal Leu-Ile zipper motif remained very rigid (<1.5 Å RMSD), while its C-terminal polar region was dynamic and splayed open forming a water-filled pore. Our engineered variant PL5 (50% similarity) has an extended Leu-Ile zipper to replace the C-terminal polar region. In simulation, the now apolar C-terminus was rigidified and maintained the narrow dehydrated pore as previously observed. We showed PL5 is an alpha-helical coiled-coil pentamer in solution by analytical ultracentrifugation and CD spectroscopy in micelles. We then solved the X-ray crystal of PL5 in C8E4 micelles (3.17 Å), which is in striking agreement to the design model: 1.16 Å RMSD. Our data shows PL5 is a reliable model for phospholamban and the Leu-Ile zipper motif, and suggests phospholamban forms a dehydrated channel intermediate in size between the two reported NMR models unlikely to conduct ions.

Upside: A New Dynamics Methods Capable of Cooperative De Novo Protein Folding in CPU-Hours

The traditional trade-off in simulations between accuracy and speed is predicated on the assumption that force fields are typically well-parameterized. We re-examine this trade-off and show that a properly formulated coarse-grain model trained using machine learning methods can rival all-atom models for de novo protein folding and dynamics simulations. We use the contrastive divergence technique to train the force field directly from short simulation trajectories of 450 proteins beginning near their native structures. This method is applied to our recently developed Upside model, where the free energy for side chains are rapidly calculated at every time-step, allowing for a smooth energy landscape without steric rattling of the side chains1. After this contrastive divergence training, the model is able to fold proteins up to ∼100 residues de novo on a single core in CPU core-days2. The Upside model's success argues that simpler models that can be globally parameterized can rival more detailed but slower models whose parameterization is more challenging - more complexity does not necessarily equate to higher accuracy! Upside's ready generation of Boltzmann ensembles allows for a wide range of studies of protein folding, dynamics and binding. Additionally, in studies that incorporate experimental or bioinformatics data, including contact predictions, Upside provides an inexpensive Bayesian prior distribution that may be updated using experimental information. 1Rapid calculation of side chain packing and free energy with applications to protein molecular dynamics. https://arxiv.org/abs/1610.07277 2Trajectory-Based Parameterization of a Coarse-Grained Forcefield for High-Throughput Protein Simulation. http://www.biorxiv.org/content/early/2017/07/27/169326

Concurrent presence of on- and off-pathway folding intermediates of apoflavodoxin at physiological ionic strength.

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.

Disruptions in the crystallinity of poly(lauryl methacrylate) due to adsorption on silica

ABSTRACTThe effects of adsorption of poly(lauryl methacrylate) (PLMA), a side‐chain crystalline polymer, on silica were investigated. Fourier transform infrared spectroscopy and differential scanning calorimetry (DSC) measurements were made on both bulk and adsorbed PLMA. The reversible heat flow rates were observed as a function of temperature and the degree of crystallinity of the samples determined based on the broad melting transitions of the side chains in the surface samples. It was found that adsorption caused a disruption of the side‐chain crystallinity primarily in the tightly bound layer of the polymer, but did not significantly affect its glass transition temperature. A change in the packing of the hydrophobic side chains, as a result of adsorption, was also observed for the tightly adsorbed polymer. These results indicated that PLMA side chains in proximity to the silica surface have different properties from those in bulk PLMA. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018, 56, 89–96

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.

An Alternatively Packed Dry Molten Globule-like Intermediate in the Native State Ensemble of a Multidomain Protein.

It has been difficult to quantify the degree of side-chain conformational heterogeneity in the native (N) state ensemble of proteins and the relative energetic contributions of the side-chain packing and the hydrophobic effect in protein stability. Here, we show using multiple site-specific spectroscopic probes and tools of thermodynamics that the N state ensemble of a multidomain protein contains an equilibrium intermediate (I) whose interdomain region resembles a dry molten globule. In the I state, a tryptophan residue in the interdomain region is alternatively packed, but its secondary structure and intradomain packing are N-like. The I state also has a larger interdomain distance, but the domain-domain interface is dry and molten. Our results indicate that hydrophobic desolvation and side-chain packing are decoupled during protein folding and that interdomain packing interactions have an important energetic contribution in protein stability. Dynamic interconversion between alternatively packed N-like states could be important for multiple allosteric and ligand binding functions of this protein.

Conformational landscape mapping the difference between N-lobes and C-lobes of calmodulin

Calmodulin is a calcium binding protein that consists of four EF-hand domains. The two EF-lobes of calmodulin, called the N-lobe and the C-lobe, arose from duplication and fusion of a precursor EF-hand. The amino acid sequences and the structures of the N-lobe and of the C-lobe are quite similar to each other. The N-lobe and the C-lobe, however, have subtle differences in structure and function. We analyzed the helix positions of calmodulin lobes by the alignment with the pseudo-two fold axis of the EF-lobe. We made a map of conformational landscape of helix positions. The four states of the EF-lobe appeared on two lines in the landscape; these two lines show the trajectory of opening and closing of the EF-lobe. For the N-lobe of calmodulin, the calcium bound form and the apo-forms are on the lower line. The two apo-forms of the C-lobe of calmodulin, with target and without target, are on the upper line. The calcium bound form of the C-lobe is on the lower line. The rearrangement of helix interaction between two the EF-hands is necessary for calcium binding in the C-lobe. The hydrophobic packing in the apo-form of the N-lobe is similar to the packing of the N- and C-lobes of the calcium bound form. However, the packing of C-lobe side chains in the apo-form is different from these other three structures. Our detailed analysis should serve as an example that can be applied to other proteins that undergo changes in conformation upon binding effectors.

OPUS-DOSP: A Distance- and Orientation-Dependent All-Atom Potential Derived from Side-Chain Packing

We report a new distance- and orientation-dependent, all-atom statistical potential derived from side-chain packing, named OPUS-DOSP, for protein structure modeling. The framework of OPUS-DOSP is based on OPUS-PSP, previously developed by us [JMB (2008), 376, 288–301], with refinement and new features. In particular, distance or orientation contribution is considered depending on the range of contact distance. A new auxiliary function in energy function is also introduced, in addition to the traditional Boltzmann term, in order to adjust the contributions of extreme cases. OPUS-DOSP was tested on 11 decoy sets commonly used for statistical potential benchmarking. Among 278 native structures, 239 and 249 native structures were recognized by OPUS-DOSP without and with the auxiliary function, respectively. The results show that OPUS-DOSP has an increased decoy recognition capability comparing with those of other relevant potentials to date.

Role of CysI-CysIII Disulfide Bond on the Structure and Activity of α-Conotoxins at Human Neuronal Nicotinic Acetylcholine Receptors.

α-Conotoxins preferentially antagonize muscle and neuronal nicotinic acetylcholine receptors (nAChRs). Native α-conotoxins have two disulfide links, CI–CIII and CII–CIV, and owing to the inherent properties of disulfide bonds, α-conotoxins have been systematically engineered to improve their chemical and biological properties. In this study, we explored the possibility of simplifying the disulfide framework of α-conotoxins Vc1.1, BuIA, ImI, and AuIB, by introducing [C2H,C8F] modification to the CI–CIII bond. We therefore explored the possibility of using hydrophobic packing of standard amino acid side chains to replace disulfide bonds as an alternative strategy to nonnatural amino acid cross-links. The impact of CI–CIII disulfide bond replacement on the conformation of the α-conotoxins was investigated using molecular dynamics (MD) simulations and nuclear magnetic resonance chemical shift index study. Two-electrode voltage clamp techniques and MD simulations were used to study the impact of disulfide bond deletion on the activities of the peptides at human neuronal nAChRs. All disulfide-deleted variants except ImI[C2H,C8F] had reduced potency for inhibiting nAChRs. Our results suggest that the CI–CIII disulfide bond is important to stabilize the secondary structure of α-conotoxins as well as their interaction with neuronal nAChR targets. Results from this study enrich our understanding of the function of the CI–CIII disulfide bond and are useful in guiding future structural engineering of the α-conotoxins.

Interpretation of Seemingly Contradictory Data: Low NMR S2 Order Parameters Observed in Helices and High NMR S2 Order Parameters in Disordered Loops of the Protein hGH at Low pH.

At low pH, human growth hormone (hGH) adopts a partially folded state, in which the native helices are maintained, but the long loop regions and side-chain packing become disordered. Some of the S2 order parameters for backbone N-H vectors derived from NMR relaxation measurements on hGH at low pH initially seem contradictory. Three isolated residues (15, 20, and 171) in helices A and D exhibit low order parameter values (<0.5) indicating flexibility, whereas residue 143 in the centre of a long flexible loop region has a high order parameter (0.82). Using S2 order parameter restraining MD simulations, this paradox has been resolved. Low S2 values in helices are due to the presence of a mixture of 310 -helical and α-helical hydrogen bonds. High S2 values in relatively disordered parts of a protein may be due to fluctuating networks of hydrogen bonds between the backbone and the side chains, which restrict the motion of N-H bond vectors.

Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints

Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron–electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3–60Å while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.

A Versatile Method to Fabricate Highly In‐Plane Aligned Conducting Polymer Films with Anisotropic Charge Transport and Thermoelectric Properties: The Key Role of Alkyl Side Chain Layers on the Doping Mechanism

A general method is proposed to produce oriented and highly crystalline conducting polymer layers. It combines the controlled orientation/crystallization of polymer films by high‐temperature rubbing with a soft‐doping method based on spin‐coating a solution of dopants in an orthogonal solvent. Doping rubbed films of regioregular poly(3‐alkylthiophene)s and poly(2,5‐bis(3‐dodecylthiophen‐2‐yl)thieno[3,2‐b]thiophene) with 2,3,5,6‐tetrafluoro‐7,7,8,8‐tetracyanoquinodimethane (F4TCNQ) yields highly oriented conducting polymer films that display polarized UV–visible–near‐infrared (NIR) absorption, anisotropy in charge transport, and thermoelectric properties. Transmission electron microscopy and polarized UV–vis–NIR spectroscopy help understand and clarify the structure of the films and the doping mechanism. F4TCNQ− anions are incorporated into the layers of side chains and orient with their long molecular axis perpendicular to the polymer chains. The ordering of dopant molecules depends closely on the length and packing of the alkyl side chains. Increasing the dopant concentration results in a continuous variation of unit cell parameters of the doped phase. The high orientation results in anisotropic charge conductivity (σ) and thermoelectric properties that are both enhanced in the direction of the polymer chains (σ = 22 ± 5 S cm−1 and S = 60 ± 2 µV K−1). The method of fabrication of such highly oriented conducting polymer films is versatile and is applicable to a large palette of semiconducting polymers.

Interrelations Between Side Chain and Main Chain Packing in Different Crystal Modifications of Alkoxylated Polyesters

Interrelations between main chain and side chain packing are studied in a series of comb-like poly(1,4-phenylene-2,5-n-dialkyloxy terephthalate)s (PPAOTs) with C = 6-12 alkyl carbons per side chain by X-ray diffractometry. Two different polymorphic states, called modification A and modification B, are observed depending on thermal treatment and side chain length. For PPAOTs with short side chains (C ≤ 8), modification A is commonly observed. "As synthesized" (solution crystallized) PPAOTs with longer side chains (C ≥ 10) contain mostly modification B while modification A is growing during melt cooling. A solid-solid transition from modification B to modification A is observed above ≈70 °C for the decyl member (C = 10, PPDOT) while modification A converts under ambient conditions slowly to modification B. This indicates that modification B is thermodynamically stable at low temperature while modification A is stable at higher temperature. Crystallographic analysis shows that both modifications are characterized by an orthorhombic unit cell and a long-range ordered layered structure with alternating main chain and alkyl nanodomains. This is confirmed by 2D diffration data for shear oriented PPDOT fibers. The main difference between both modifications is the packing of the side chains which are in a crystalline state for modification B but disordered for modification A. This is concluded from values for the volume per CH2 unit VCH2 in alkyl nanodomains calculated without further assumptions from the obtained lattice parameters. Interestingly, the crystalline packing of the side chains in modification B leads to a significant increase (≈ 20%) in the application relevant π-π spacings within the main chain domains as compared to those for modification A. It is argued that the structure formation process and thermodynamic equilibrium in comb-like polymers might be strongly influenced by a competition of the individual packing tendencies of main and side chains.

Modes of Cholesterol Binding Influence the Interaction of Syrian Hamster PrPC with the Plasma Membrane

Prions are infectious agents responsible for transmissible spongiform encephalopathies, a fatal neurodegenerative disease in mammals, including humans. Prions propagate biological information by conversion of the non pathological version of the prion protein PrPc to the infectious isoform, PrPSc. Normally, the cellular prion protein, PrPc, is GPI-anchored to the outer leaflet of the cell membrane. There is evidence that this attachment occurs at membrane domains called lipid rafts, which are rich in cholesterol. Furthermore, PrPSc propagation and associated infectivity have been proven to be reduced by downregulation of cholesterol uptake, export, and complexation, and to interfere with cholesterol metabolism. Therefore, we conducted a structural bioinformatics study to understand the interactions between PrPc and cholesterol molecules at the atomistic level. Our free energy calculations suggest that the modes of binding of cholesterol molecules to the surface of the Syrian hamster PrPc modulate the interaction between PrPc and model plasma membranes. Such modulation depends on local cholesterol-PrPc side chain packing and optimal accommodation of the cholesterol hydroxyl group.

Peptide Membrane Displaying Membrane Fusion and Fission with Lipid–Raft–Like Organization

Organelles in cells take a wide range of morphologies and convert one morphology to the other for specific biological events. We are trying to construct such complex morphologies allowing transformation from one to the other by using molecular assemblies comprising amphiphilic helical peptides. One key player is poly(sarcosine)–b–(L–Leu- Aib)n having a hydrophilic block of poly(sarcosine) and a helical hydrophobic block of (L–Leu–Aib)n. The driving forces for making these molecular assemblies can be modulated widely by helical block length, knobs-into-holes packing of side chains between helix surfaces, and stereocomplex formation between right- and left–handed helices. Under properly designed amphiphilic polypeptides and their combinations, we can construct morphologies of sheet, tube, and vesicles and even the combination of tube and vesicle, a round–bottomed flask shape. There were several combinations of amphiphilic polypeptides to take the round–bottomed flask morphology. The chimeric morphology is a direct consequence of phase separation of the component polypeptides in one consecutive membrane. The distribution of the components into the round–bottomed or neck moiety was dependent on the combination meaning that lateral diffusion of the components in the membrane led to the phase separation. The vesicle composed of poly(sarcosine)– b–(L–Leu–Aib)6 and poly(sarcosine)–b–(D–Leu–Aib)6 allowed membrane fusion and fission above the phase transition temperature with itself and other vesicle. The monolayer elasticity of vesicles was variable in accordance with the component amphiphilic polypeptides, and the elasticity similar to liposome can be prepared.

PdII2L4-type coordination cages up to three nanometers in size.

The utilization of large ligands in coordination-based self-assembly represents an attractive strategy for the construction of supramolecular assemblies more than two nanometers in size. However, the implementation of this strategy is hampered by the fact that the preparation of such ligands often requires substantial synthetic effort. Herein, we describe a simple one-step protocol, which allows large bipyridyl ligands with a bent shape to be synthesized from easily accessible and/or commercially available starting materials. The ligands were used to construct PdII2L4-type coordination cages of unprecedented size. Furthermore, we provide evidence that these cages may be stabilized by close intramolecular packing of lipophilic ligand side chains. Packing effects of this kind are frequently encountered in protein assemblies, but they are seldom used as a design element in metallasupramolecular chemistry.

Multiple Chain Packing and Phase Composition in Regioregular Poly(3-butylthiophene) Films

On the basis of different film preparation conditions, we address the phase composition and thiophene main-chain and butyl side-chain organization of three regioregular poly(3-butylthiophene) (P3BT) samples prepared by drop-casting. These samples are readily identified by wide-angle X-ray diffraction (WAXD) with the structural P3BT polymorphs commonly referred to as forms I, I?, and II. Here, we show by means of WAXD, solid-state nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR) that the P3BT samples identified with these polymeric forms (I, I?, and II) do not contain a single, unique crystalline phase and an amorphous phase, but additionally consist of one (form II) or two (forms I and I?) distinct crystalline phases. Each of the crystalline phases is associated with specific thiophene main-chain packing arrangements and butyl side-chain organizations. We provide a spectroscopic identification for each of the P3BT crystalline phases, enabling a detailed and consistent picture of molecular order and polymer main-chain and side-chain packing. Thus, our results are expected to be valuable in future studies of poly(3-alkylthiophene) (P3AT) thin films, where in particular FTIR studies will permit quick and easy access to both phase composition and identification of multiple thiophene main-chain packing structures.

Effects of Backbone Planarity and Tightly Packed Alkyl Chains in the Donor–Acceptor Polymers for High Photostability

The photostability of donor–acceptor (D–A) polymers remains a critical issue despite recent improvements in the power conversion efficiencies (PCEs) of organic photovoltaic (OPV) cells. We report the synthesis of three highly photostable polymers (PDTBDT-BZ, PDTBDT-BZF, and PDTBDT-BZF2) and their suitability for use in high-performance OPV cells. Under 1 sunlight of illumination in air for 10 h, these polymer films demonstrated remarkably high photostability compared to that of PTB7, a representative polymer in the OPV field. While the PDTBDT-BZ, PDTBDT-BZF, and PDTBDT-BZF2 polymer films maintained 97, 90, and 96% photostability, respectively, a PTB7 film exhibited only 38% photostability under the same conditions. We ascribed the high photostability of the polymers to both the intrinsically photostable chemical moieties and the dense packing of alkyl side chains and planar backbone polymer chains, which prevents oxygen diffusion into the PDTBDT-BZ films. This work demonstrates the high photostability of planar PDTBDT-BZ series polymers composed of photostable DTBDT and BZ moieties and suggests a design rule to synthesize highly photostable photovoltaic materials.

Protein side-chain packing problem: is there still room for improvement?

The protein side-chain packing problem (PSCPP) is an important subproblem of both protein structure prediction and protein design. During the past two decades, a large number of methods have been proposed to tackle this problem. These methods consist of three main components: a rotamer library, a scoring function and a search strategy. The average overall accuracy level obtained by these methods is approximately 87%. Whether a better accuracy level could be achieved remains to be answered. To address this question, we calculated the maximum accuracy level attainable using a simple rotamer library, independently of the energy function or the search method. Using 2883 different structures from the Protein Data Bank, we compared this accuracy level with the accuracy level of five state-of-the-art methods. These comparisons indicated that, for buried residues in the protein, we are already close to the best possible accuracy results. In addition, for exposed residues, we found that a significant gap exists between the possible improvement and the maximum accuracy level achievable with current methods. After determining that an improvement is possible, the next step is to understand what limitations are preventing us from obtaining such an improvement. Previous works on protein structure prediction and protein design have shown that scoring function inaccuracies may represent the main obstacle to achieving better results for these problems. To show that the same is true for the PSCPP, we evaluated the quality of two scoring functions used by some state-of-the-art algorithms. Our results indicate that neither of these scoring functions can guide the search method correctly, thereby reinforcing the idea that efforts to solve the PSCPP must also focus on developing better scoring functions.