A solution structure analysis reveals a bent collagen triple helix in the complement activation recognition molecule mannan-binding lectin.

Collagen triple helices are critical in the function of mannan-binding lectin (MBL), an oligomeric recognition molecule in complement activation. The MBL collagen regions form complexes with the serine proteases MASP-1 and MASP-2 in order to activate complement, and mutations lead to common immunodeficiencies. To evaluate their structure-function properties, we studied the solution structures of four MBL-like collagen peptides. The thermal stability of the MBL collagen region was much reduced by the presence of a GQG interruption in the typical (X-Y-Gly)n repeat compared to controls. Experimental solution structural data were collected using analytical ultracentrifugation and small angle X-ray and neutron scattering. As controls, we included two standard Pro-Hyp-Gly collagen peptides (POG)10-13, as well as three more peptides with diverse (X-Y-Gly)n sequences that represented other collagen features. These data were quantitatively compared with atomistic linear collagen models derived from crystal structures and 12,000 conformations obtained from molecular dynamics simulations. All four MBL peptides were bent to varying degrees up to 85o in the best-fit molecular dynamics models. The best-fit benchmark peptides (POG)n were more linear but exhibited a degree of conformational flexibility. The remaining three peptides showed mostly linear solution structures. In conclusion, the collagen helix is not strictly linear, the degree of flexibility in the triple helix depends on its sequence, and the triple helix with the GQG interruption showed a pronounced bend. The bend in MBL GQG peptides resembles the bend in the collagen of complement C1q and may be key for lectin pathway activation.

Volumetric Properties of the Transition State Ensemble for Protein Folding.

Understanding how high hydrostatic pressure affects biomacromolecular interaction is important for deciphering the molecular mechanisms by which organisms adapt to live at the bottom of the ocean. The relative effect of hydrostatic pressure on the rates of folding/unfolding reactions is defined by the volumetric properties of the transition state ensemble relative to the folded and unfolded states. All-atom structure-based molecular dynamics simulations combined with quantitative computational protocol to compute volumes from three-dimensional coordinates allow volumetric mapping of protein folding landscape. This, is turn, provides qualitative understanding of the effects of hydrostatic pressure on energy landscape of proteins. The computational results for six different proteins are directly benchmark against experimental data and show an excellent agreement. Both experiments and computation show that the transition-state ensemble volume appears to be in-between the folded and unfolded state volumes, and thus the hydrostatic pressure accelerates protein unfolding.

The volume changes of unfolding of dsDNA.

High hydrostatic pressure can have profound effects on the stability of biomacromolecules. The magnitude and direction (stabilizing or destabilizing) of this effect is defined by the volume changes in the system, ΔV. Positive volume changes will stabilize the starting native state, whereas negative volume changes will lead to the stabilization of the final unfolded state. For the DNA double helix, experimental data suggested that when the thermostability of dsDNA is below 50°C, increase in hydrostatic pressure will lead to destabilization; i.e., helix-to-coil transition has negative ΔV. In contrast, the dsDNA sequences with the thermostability above 50°C showed positive ΔV values and were stabilized by hydrostatic pressure. In order to get insight into this switch in the response of dsDNA to hydrostatic pressure as a function of temperature, first we further validated this trend using experimental measurements of ΔV for 10 different dsDNA sequences using pressure perturbation calorimetry. We also developed a computational protocol to calculate the expected volume changes of dsDNA unfolding, which was benchmarked against the experimental set of 50 ΔV values that included, in addition to our data, the values from the literature. Computation predicts well the experimental values of ΔV. Such agreement between computation and experiment lends credibility to the computation protocol and provides molecular level rational for the observed temperature dependence of ΔV that can be traced to the hydration. Difference in the ΔV value for A/T versus G/C basepairs is also discussed.

From Protein Design to the Energy Landscape of a Cold Unfolding Protein.

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

Effects of Hydrostatic Pressure on the Thermodynamics of CspB-Bs Interactions with the ssDNA Template.

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔGProto, the volume change upon CspB-Bs unfolding, ΔVProt, the association constant for CspB-Bs:pDT7 complex, Kao, and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔVBind. The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔVProt < 0). Surprisingly, our study showed that ΔVBind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein-ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein-ssDNA complex under given conditions.

Sequence-independent recognition of the amyloid structural motif by GFP protein family.

Cnidarian fluorescent protein (FP) derivatives such as GFP, mCherry, and mEOS2 have been widely used to monitor gene expression and protein localization through biological imaging because they are considered functionally inert. We demonstrate that FPs specifically bind amyloid fibrils formed from many natural peptides and proteins. FPs do not bind other nonamyloid fibrillar structures such as microtubules or actin filaments and do not bind to amorphous aggregates. FPs can also bind small aggregates formed during the lag phase and early elongation phase of fibril formation and can inhibit amyloid fibril formation in a dose-dependent manner. These findings suggest caution should be taken in interpreting FP-fusion protein localization data when amyloid structures may be present. Given the pathological significance of amyloid-related species in some diseases, detection and inhibition of amyloid fibril formation using FPs can provide insights on developing diagnostic tools.

Evolutionary conservation and structural localizations suggest a physical trace of metabolism's progressive geochronological emergence.

We studied multiple sequence alignment (MSA) consensus amino acid distributional patterns in 2844 amino acid sequences of the eight enzymes of the Kreb's oxidative tricarboxylic acid pathway (oTCA) in Archaea, Bacteria and Eukarya and 5545 sequences of 33 bacteria as geochronologically separated enzymes with MSA consensus site modal identities. The 33 bacteria were 20 presumptive examples of early-oldest (Hadean-Archaean) ('Epoch I') or 13 late-newest (contemporary) ('Epoch III') appearing enzymes on Earth. The enzyme's MSA consensus sites were identified by their modal identity, % Occupancy in one of nine-graded evolutionary-conservation zones (CZs) and the Euclidean distance (Å) from each of their consensus MSA Cɑs to the same atom (Anchor-atom) in their reported functional center. These MSA consensus sites are tetrad-data points called recovered-amino acids (RAA). Across Domains, the % Occupancies of the eight-dominant RAAs of the Kreb's cycle and the 33 bacteria were found to be similarly ranked. Compared to Trifonov's 'putative ranked temporal order of the appearance of amino acids on Earth' (TOAE), the greatest statistical concordance with tetrad-RAAs across Domains were those characterized as within the most-evolutionary conserved conservation zone (CZ9), typically nearest (Å) their enzyme's catalytic/active center. The geochronologically characterized early-oldest Hadean-Archaean Bacteria 'Epoch I' enzymes, compared to late-newest Bacteria enzymes, had greater average numbers of amino acid residues/sequence and a statistically significant larger variability in their RAA compositional-Å3-volumes. The late-newest 'Epoch III' enzymes had statistically significant lower volumetric values, specifically, their native Å3-volume, void-volume and volume change on unfolding. Our enzyme data suggest a geochronological trace of 'metabolism's progressive emergence'.Communicated by Ramaswamy H. Sarma.

Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome.

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔVTot ) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔVTot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔVTot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔVTot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.

Predictive Model of Linear Antimicrobial Peptides Active against Gram-Negative Bacteria.

Antimicrobial peptides (AMPs) have been identified as a potential new class of anti-infectives for drug development. There are a lot of computational methods that try to predict AMPs. Most of them can only predict if a peptide will show any antimicrobial potency, but to the best of our knowledge, there are no tools which can predict antimicrobial potency against particular strains. Here we present a predictive model of linear AMPs being active against particular Gram-negative strains relying on a semi-supervised machine-learning approach with a density-based clustering algorithm. The algorithm can well distinguish peptides active against particular strains from others which may also be active but not against the considered strain. The available AMP prediction tools cannot carry out this task. The prediction tool based on the algorithm suggested herein is available on https://dbaasp.org.

Strategy for Stabilization of CutA1 Proteins Due to Ion-Ion Interactions at Temperatures of over 100 °C.

In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 °C, we constructed many mutants of the CutA1 protein ( EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 °C. The hydrophobic mutant of EcCutA1 ( Ec0VV) with denaturation temperature ( Td) of 113.2 °C was used as a template for mutations. The highest Td of Ec0VV mutants substituted by a single charged residue was 118.4 °C. Multiple ion mutants were also constructed by combination of single mutants and found to have an increased thermostability. The highest stability of multiple mutants was a mutant substituted by nine charged residues that had a Td of 142.2 °C. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by a molecular dynamics simulation at 300 K. The Td of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 °C, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.

Molecular Determinants of Temperature Dependence of Protein Volume Change upon Unfolding.

Pressure is a well-known environmental stressor that can either stabilize or destabilize proteins. The volumetric change upon protein unfolding determines the effect of pressure on protein stability, where negative volume changes destabilized proteins at high pressures. High temperature often accompanies high pressure, for example, in the ocean depths near hydrothermal vents or near faults, so it is important to study the effect of temperature on the volumetric change upon unfolding. We previously detailed the magnitude and sign of the molecular determinants of volumetric change, allowing us to quantitatively predict the volumetric change upon protein unfolding. Here, we present a comprehensive analysis of the temperature dependence of the volumetric components of proteins, showing that hydration volume is the primary component that defines expansivities of the native and unfolded states and void volume only contributes slightly to the folded state expansivity.

Putting the Piezolyte Hypothesis under Pressure.

A group of small molecules that stabilize proteins against high hydrostatic pressure has been classified as piezolytes, a subset of stabilizing cosolutes. This distinction would imply that piezolytes counteract the effects of high hydrostatic pressure through effects on the volumetric properties of the protein. The purpose of this study was to determine if cosolutes proposed to be piezolytes have an effect on the volumetric properties of proteins through direct experimental measurements of volume changes upon unfolding of model proteins lysozyme and ribonuclease A, in solutions containing varying cosolute concentrations. Solutions containing the proposed piezolytes glutamate, sarcosine, and betaine were used, as well as solutions containing the denaturants guanidinium hydrochloride and urea. Changes in thermostability were monitored using differential scanning calorimetry whereas changes in volume were monitored using pressure perturbation calorimetry. Our findings indicate that increasing stabilizing cosolute concentration increases the stability and transition temperature of the protein, but does not change the temperature dependence of volume changes upon unfolding. The results suggest that the pressure stability of a protein in solution is not directly affected by the presence of these proposed piezolytes, and so they cannot be granted this distinction.

Non-linearity of the collagen triple helix in solution and implications for collagen function.

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly)n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG)10unblocked, was compared with the blocked (POG)10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4-17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease.

Evidence for the principle of minimal frustration in the evolution of protein folding landscapes.

Theoretical and experimental studies have firmly established that protein folding can be described by a funneled energy landscape. This funneled energy landscape is the result of foldable protein sequences evolving following the principle of minimal frustration, which allows proteins to rapidly fold to their native biologically functional conformations. For a protein family with a given functional fold, the principle of minimal frustration suggests that, independent of sequence, all proteins within this family should fold with similar rates. However, depending on the optimal living temperature of the organism, proteins also need to modulate their thermodynamic stability. Consequently, the difference in thermodynamic stability should be primarily caused by differences in the unfolding rates. To test this hypothesis experimentally, we performed comprehensive thermodynamic and kinetic analyses of 15 different proteins from the thioredoxin family. Eight of these thioredoxins were extant proteins from psychrophilic, mesophilic, or thermophilic organisms. The other seven protein sequences were obtained using ancestral sequence reconstruction and can be dated back over 4 billion years. We found that all studied proteins fold with very similar rates but unfold with rates that differ up to three orders of magnitude. The unfolding rates correlate well with the thermodynamic stability of the proteins. Moreover, proteins that unfold slower are more resistant to proteolysis. These results provide direct experimental support to the principle of minimal frustration hypothesis.

Linking computation and experiments to study the role of charge-charge interactions in protein folding and stability.

Over the past two decades there has been an increase in appreciation for the role of surface charge-charge interactions in protein folding and stability. The perception shifted from the belief that charge-charge interactions are not important for protein folding and stability to the near quantitative understanding of how these interactions shape the folding energy landscape. This led to the ability of computational approaches to rationally redesign surface charge-charge interactions to modulate thermodynamic properties of proteins. Here we summarize our progress in understanding the role of charge-charge interactions for protein stability using examples drawn from my own laboratory and touch upon unanswered questions.

Molecular determinant of the effects of hydrostatic pressure on protein folding stability.

Hydrostatic pressure is an important environmental variable that plays an essential role in biological adaptation for many extremophilic organisms (for example, piezophiles). Increase in hydrostatic pressure, much like increase in temperature, perturbs the thermodynamic equilibrium between native and unfolded states of proteins. Experimentally, it has been observed that increase in hydrostatic pressure can both increase and decrease protein stability. These observations suggest that volume changes upon protein unfolding can be both positive and negative. The molecular details of this difference in sign of volume changes have been puzzling the field for the past 50 years. Here we present a comprehensive thermodynamic model that provides in-depth analysis of the contribution of various molecular determinants to the volume changes upon protein unfolding. Comparison with experimental data shows that the model allows quantitative predictions of volume changes upon protein unfolding, thus paving the way to proteome-wide computational comparison of proteins from different extremophilic organisms.

Intramolecular diffusion controls aggregation of the PAPf39 peptide.

The 39-residue fragment of human prostatic acidic phosphatase (PAP) is found in high concentrations in semen and easily form fibrils. Previous work has shown that fibrillization is accelerated with a deletion of the first 8, mostly charged residues and it was hypothesized that fibrillization depended on the dynamics of these peptides. To test this hypothesis we have measured the intramolecular diffusion of the full length and 8-residue deletion peptides at two different pHs and found a correlation with fibrillization lag time. These results can be explained by a simple kinetic model of the early stages of aggregation in which oligomerization is controlled by the rate of peptide reconfiguration.

Gallic Acid Is an Antagonist of Semen Amyloid Fibrils That Enhance HIV-1 Infection.

Recent in vitro studies have demonstrated that amyloid fibrils found in semen from healthy and HIV-infected men, as well as semen itself, can markedly enhance HIV infection rates. Semen fibrils are made up of multiple naturally occurring peptide fragments derived from semen. The best characterized of these fibrils are SEVI (semen-derived enhancer of viral infection), made up of residues 248-286 of prostatic acidic phosphatase, and the SEM1 fibrils, made up of residues 86-107 of semenogelin 1. A small molecule screen for antagonists of semen fibrils identified four compounds that lowered semen-mediated enhancement of HIV-1 infectivity. One of the four, gallic acid, was previously reported to antagonize other amyloids and to exert anti-inflammatory effects. To better understand the mechanism by which gallic acid modifies the properties of semen amyloids, we performed biophysical measurements (atomic force microscopy, electron microscopy, confocal microscopy, thioflavin T and Congo Red fluorescence assays, zeta potential measurements) and quantitative assays on the effects of gallic acid on semen-mediated enhancement of HIV infection and inflammation. Our results demonstrate that gallic acid binds to both SEVI and SEM1 fibrils and modifies their surface electrostatics to render them less cationic. In addition, gallic acid decreased semen-mediated enhancement of HIV infection but did not decrease the inflammatory response induced by semen. Together, these observations identify gallic acid as a non-polyanionic compound that inhibits semen-mediated enhancement of HIV infection and suggest the potential utility of incorporating gallic acid into a multicomponent microbicide targeting both the HIV virus and host components that promote viral infection.

Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding.

BACKGROUND: Pressure perturbation calorimetry (PPC) is a biophysical method that allows direct determination of the volume changes upon conformational transitions in macromolecules. SCOPE OF THIS REVIEW: This review provides novel details of the use of PPC to analyze unfolding transitions in proteins. The emphasis is made on the data analysis as well as on the validation of different structural factors that define the volume changes upon unfolding. Four case studies are presented that show the application of these concepts to various protein systems. MAJOR CONCLUSIONS: The major conclusions are: 1. Knowledge of the thermodynamic parameters for heat induced unfolding facilitates the analysis of the PPC profiles. 2. The changes in the thermal expansion coefficient upon unfolding appear to be temperature dependent.3.Substitutions on the protein surface have negligible effects on the volume changes upon protein unfolding. 4. Structural plasticity of proteins defines the position dependent effect of amino acid substitutions of the residues buried in the native state. 5. Small proteins have positive volume changes upon unfolding which suggests difference in balance between the cavity/void volume in the native state and the hydration volume changes upon unfolding as compared to the large proteins that have negative volume changes. GENERAL SIGNIFICANCE: The information provided here gives a better understanding and deeper insight into the role played by various factors in defining the volume changes upon protein unfolding.