Glycine rich segments adopt polyproline II helices: Implications for biomolecular condensate formation.

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Based on NMR studies of the Gly-rich H. harveyi "snow flea" antifreeze protein, we recently proposed that Gly-rich sequences, such as the third "RGG" region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.

Depressive symptoms and behavioural health risks in young women attending an urban sexual health clinic.

BACKGROUND: Adolescence is a time of increased health risk behaviours and depressive symptoms and disorders. Most young people with depressive disorders, however, are not under the care of specialist child and adolescent mental health services, and there is increasing interest in identifying alternative appropriate settings which are acceptable for young people and may attract those at high risk for depressive disorders. AIM: To assess depressive and behavioural health risks in attendees to a sexual health clinic (SHC) for young people. METHOD: Cross-sectional controlled study of women attending a SHC and school attendees, using questionnaires on depressive symptoms and mental health problems, sexual activity and substance use. RESULTS: 115/178 attendees 14-19 years old completed questionnaires. In the comparison between 66 women aged 16-18 years and 49 socio-demographically group-matched school controls, SHC attendees reported higher levels of sexual activity and substance use. They were significantly more at risk for depression (20/65 or 30% vs. 4/46 or 8%; chi(2)P < 0.01) on the Beck Depression Inventory, for emotional (12/66 or 18% vs. 1/49 or 2%; chi(2)P < 0.01), hyperactivity (11/66 or 16% vs. 2/46 or 4%; chi(2)P < 0.05) and conduct (8/66 or 12% vs. 0/49; chi(2)P < 0.05) problems on the Strengths and Difficulties Questionnaire. High depressive scores were significantly associated with a history of self-harm, earlier alcohol use and having tried cannabis, but not with sexual risk behaviours. Earlier age at first sexual intercourse was linked to higher sexual and substance use risks. CONCLUSION: We have found high levels of depressive symptoms among attendees to a SHC for young people, which seem an appropriate setting for screening and providing guidance for depression and other health risk behaviours.

Studies of the aggregation of an amyloidogenic alpha-synuclein peptide fragment.

The deposition of alpha-syn (alpha-synuclein) fibrils in Lewy bodies is a characteristic feature of individuals with neurodegenerative disorders. A peptide comprising the central residues 71-82 of alpha-syn [alpha-syn(71-82)] is capable of forming beta-sheet-rich, amyloid-like fibrils with similar morphologies to fibrils of the full-length protein, providing a useful model of pathogenic alpha-syn fibrils that is suitable for detailed structural analysis. We have studied the morphology and gross structural features of alpha-syn(71-82) fibrils formed under different conditions in order to obtain reliable conditions for producing fibrils for further structural investigations. The results indicate that the rate of aggregation and the morphology of the fibrils formed are sensitive to pH and temperature.

Impaired parasympathetic response to feeding in ventilated preterm babies.

BACKGROUND: Premature very low birthweight (VLBW) infants are born with an underdeveloped parasympathetic nervous system (PNS) which may limit their ability to respond adequately to feeding and may limit their capacities for extrauterine growth and development. OBJECTIVES: To describe the patterns of autonomic response to feeding and identify relationships between change in heart period variability measures over time with selected infant characteristics. METHODS: Individual growth curve analysis techniques were used to describe the patterns of change over time in sympathetic and parasympathetic tone as measured by low and high frequency heart period power. RESULTS: Sixteen mechanically ventilated VLBW infants with a mean corrected gestational age of 30.4 weeks participated in the study. The low frequency (LF) power slope was -17.67 (p = 0.0002) and the high frequency (HF) power slope was -0.92 (0.0003). There was a significant relationship between HF slope and birth gestational age (r = -0.49, p = 0.05). CONCLUSIONS: HF power, representing primarily parasympathetic activity, did not increase with enteral feeding as anticipated. LF power, an indicator of sympathetic tone, decreased during and after feeding suggesting the anticipated effect of inhibition of the sympathetic nervous system in response to the gut stimulus. Critically ill VLBW infants possess an overriding sympathetic response, but may not have adequate PNS tone development.

The aggregation and membrane-binding properties of an alpha-synuclein peptide fragment.

alpha-Synuclein is a 140 amino acid protein, which is associated with presynaptic membranes in the brain, and is the major component of protein aggregates produced during the progression of many neurodegenerative diseases. It has been shown that a central hydrophobic region of alpha-synuclein comprising residues 71-82 is required for aggregation of the protein into the fibrillar form found in pathogenic aggregates [Giasson, Murray, Trojanowski and Lee (2001) J. Biol. Chem. 276, 2380-2386]. In the present study, we used (2)H NMR and electron microscopy to investigate the aggregation and membrane-binding properties of a synthetic peptide corresponding to this region. Results indicate that this region associates with phospholipid bilayers but also forms amyloid-like fibrils in the absence of lipid membranes.

Inhibition of toxicity and protofibril formation in the amyloid-beta peptide beta(25-35) using N-methylated derivatives.

Beta (25-35) is a fragment of beta-amyloid that retains its wild-type properties. N-methylated derivatives of beta(25-35) can block hydrogen bonding on the outer edge of the assembling amyloid, so preventing the aggregation and inhibiting the toxicity of the wild-type peptide. The effects are assayed by Congo Red and thioflavin T binding, electron microscopy and an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] toxicity assay. N-methyl-Gly-25 has similar properties to the wild- type, while five other methylation sites have varying effects on prefolded fibrils and fibril assembly. In particular, N-methyl-Gly-33 is able to completely prevent fibril assembly and reduces the toxicity of prefolded amyloid. With N-methyl-Leu-34 the fibril morphology is altered and toxicity reduced. A preliminary study of beta(25-35) structure in aqueous solution was made by small-angle neutron scattering (SANS). The protofibrillar aggregates are best described as a disc of radius 140 A and height 53 A (1 A = 0.1 nm), though the possibility of polydisperse aggregates cannot be ruled out. No aggregates form in the presence of N-methyl-Gly-33. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity and that SANS is an important technique for the direct observation of protofibril formation and destruction in solution.

Stabilizing nonpolar/polar side-chain interactions in the alpha-helix.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in alpha-helices, can in fact be stabilizing. Residues spaced i, i + 4 in alpha-helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile-Lys, Ile-Arg, and Val-Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix-coil theory to calculate the free energy for the interactions. All three stabilize the helix with DeltaG between -0.14 and -0.32 kcal x mol(-1). The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues.

Structure, stability and folding of the alpha-helix.

Pauling first described the alpha-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 3(10)-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or alpha L C-cap motif. The kinetics of alpha-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.

Effect of the N2 residue on the stability of the alpha-helix for all 20 amino acids.

N2 is the second position in the alpha-helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH(3)CO-[AXAAAAKAAAAKAAGY]-NH(2)) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 residue identity has been used to determine a free-energy scale by analysis with a modified Lifson-Roig helix-coil theory that includes a parameter for the N2 energy (n2). The rank order of DeltaDeltaG((relative to Ala)) is Glu(-), Asp(-) > Ala > Glu(0), Leu, Val, Gln, Thr, Ile, Ser, Met, Asp(0), His(0), Arg, Cys, Lys, Phe > Asn, > Gly, His(+), Pro, Tyr. The results correlate very well with N2 propensities in proteins, moderately well with N1 and helix interior preferences, and not at all with N-cap preferences. The strongest energetic effects result from interactions with the helix dipole, which favors negative charges at the helix N terminus. Hydrogen bonds to side chains at N2, such as Gln, Ser, and Thr, are weak, despite occurring frequently in protein crystal structures, in contrast to the N-cap position. This is because N-cap hydrogen bonds are close to linear, whereas N2 hydrogen bonds have poor geometry. These results can be used to modify protein stability rationally, help design helices, and improve prediction of helix location and stability.

Effect of the N1 residue on the stability of the alpha-helix for all 20 amino acids.

N1 is the first residue in an alpha-helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH(3)CO-XAAAAQAAAAQAAGY-NH(2) at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy. The data were analyzed by our modified Lifson-Roig helix-coil theory, which includes the n1 parameter, to find free energies for placing a residue into the N1 position. The rank order for free energies is Asp(-), Ala > Glu(-) > Glu(0) > Trp, Leu, Ser > Asp(0), Thr, Gln, Met, Ile > Val, Pro > Lys(+), Arg, His(0) > Cys, Gly > Phe > Asn, Tyr, His(+). N1 preferences are clearly distinct from preferences for the preceding N-cap and alpha-helix interior. pK(a) values were measured for Asp, Glu, and His, and protonation-free energies were calculated for Asp and Glu. The dissociation of the Asp proton is less favorable than that of Glu, and this reflects its involvement in a stronger stabilizing interaction at the N terminus. Proline is not energetically favored at the alpha-helix N terminus despite having a high propensity for this position in crystal structures. The data presented are of value both in rationalizing mutations at N1 alpha-helix sites in proteins and in predicting the helix contents of peptides.

Rotamer strain energy in protein helices - quantification of a major force opposing protein folding.

It is widely believed that the dominant force opposing protein folding is the entropic cost of restricting internal rotations. The energetic changes from restricting side-chain torsional motion are more complex than simply a loss of conformational entropy, however. A second force opposing protein folding arises when a side-chain in the folded state is not in its lowest-energy rotamer, giving rotameric strain. chi strain energy results from a dihedral angle being shifted from the most stable conformation of a rotamer when a protein folds. We calculated the energy of a side-chain as a function of its dihedral angles in a poly(Ala) helix. Using these energy profiles, we quantify conformational entropy, rotameric strain energy and chi strain energy for all 17 amino acid residues with side-chains in alpha-helices. We can calculate these terms for any amino acid in a helix interior in a protein, as a function of its side-chain dihedral angles, and have implemented this algorithm on a web page. The mean change in rotameric strain energy on folding is 0.42 kcal mol-1 per residue and the mean chi strain energy is 0.64 kcal mol-1 per residue. Loss of conformational entropy opposes folding by a mean of 1.1 kcal mol-1 per residue, and the mean total force opposing restricting a side-chain into a helix is 2.2 kcal mol-1. Conformational entropy estimates alone therefore greatly underestimate the forces opposing protein folding. The introduction of strain when a protein folds should not be neglected when attempting to quantify the balance of forces affecting protein stability. Consideration of rotameric strain energy may help the use of rotamer libraries in protein design and rationalise the effects of mutations where side-chain conformations change.

Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme.

The amyloidogenic prefibrillar partially denatured intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, was studied using Raman optical activity (ROA). A positive band in the room temperature ROA spectrum of the native protein at approximately 1345 cm(-1), assigned to a hydrated form of alpha-helix, is not present in that of the prefibrillar intermediate, where a new strong positive band at approximately 1318 cm(-1) appears instead that is assigned to the poly(l-proline) II (PPII)-helical conformation. A sharp negative band at approximately 1241 cm(-1) in the native protein, assigned to beta-strand, shows little change in the ROA spectrum of the prefibrillar intermediate. The disappearance of a positive ROA band at approximately 1551 cm(-1) assigned to vibrations of tryptophan side-chains indicates that major conformational changes have occurred among the five tryptophan residues present in human lysozyme, four of which are located in the alpha-domain. The various ROA data suggest that a substantial loss of tertiary structure has occurred in the prefibrillar intermediate and that this is located more in the alpha-domain than in the beta-domain. There is no evidence for any increase in beta-structure. The ROA spectrum of hen lysozyme, which does not form amyloid fibrils so readily, remains much more native-like on heating to 57 degrees C at pH 2.0. The thermal behaviour of the alanine-rich alpha-helical peptide AK21 in aqueous solution was found to be similar to that of human lysozyme. Hydrated alpha-helix therefore appears to readily undergo a conformational change to PPII structure on heating, which may be a key step in the conversion of alpha-helix into beta-sheet in the formation of amyloid fibrils in human lysozyme. Since it is extended, flexible, lacks intrachain hydrogen bonds and is fully hydrated in aqueous solution, PPII helix has the appropriate characteristics to be implicated as a critical conformational element in many conformational diseases. Disorder of the PPII type may be a sine qua non for the formation of regular fibrils; whereas the more dynamic disorder of the random coil may lead only to amorphous aggregates.

Determination of alpha-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory.

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.

Inhibition of toxicity in the beta-amyloid peptide fragment beta -(25-35) using N-methylated derivatives: a general strategy to prevent amyloid formation.

beta-(25-35) is a synthetic derivative of beta-amyloid, the peptide that is believed to cause Alzheimer's disease. As it is highly toxic and forms fibrillar aggregates typical of beta-amyloid, it is suitable as a model for testing inhibitors of aggregation and toxicity. We demonstrate that N-methylated derivatives of beta-(25-35), which in isolation are soluble and non-toxic, can prevent the aggregation and inhibit the resulting toxicity of the wild type peptide. N-Methylation can block hydrogen bonding on the outer edge of the assembling amyloid. The peptides are assayed by Congo red and thioflavin T binding, electron microscopy, and a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay on PC12 cells. One peptide (Gly(25) N-methylated) has properties similar to the wild type, whereas five have varying effects on prefolded fibrils and fibril assembly. In particular, beta-(25-35) with Gly(33) N-methylated is able to completely prevent fibril assembly and to reduce the toxicity of prefolded amyloid. With Leu(34) N-methylated, the fibril morphology is altered and the toxicity reduced. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity.

Periodicity in alpha-helix lengths and C-capping preferences.

We surveyed 299 high resolution, non-homologous protein crystal structures for alpha-helix lengths and capping preferences. We find that helices show a preference to have close to an integral number of turns. Helices can be usefully subdivided into either "favoured length" with 6, 7, 10, 11, 13, 14, 17, 18, 21, 22, 24, 25, 28, 29 or 31 residues, or "disfavoured length" with 8, 9, 12, 15, 16, 19, 20, 23, 26, 27 or 30 residues. Favoured length helices have their N and C-caps on the same side of the helix so they can lie on the protein surface. There is no significant difference in amino acid preferences at the N terminus between favoured and disfavoured length helices. At the C terminus, favoured length helices prefer non-polar side-chains at C4 and polar amino acid residues at C2, while disfavoured length helices prefer non-polar amino acid residues at C2. There are strong periodic trends in the likelihood of terminating a helix with a Schellman or alphaL C-capping motif. These can be rationalised by the preference for a non-polar side-chain at C3 with these motifs, favouring placing C3 on the buried side of the helix. We suggest that algorithms aiming to predict helices or C-capping in proteins should include a weight for the helix length.

The alpha-helix folds on the millisecond time scale.

It has long been believed that nucleation of the alpha-helix is a very fast reaction, occurring in around 10(-7) s. We show here that helix nucleation, in fact, takes place on the millisecond time scale. The rate of alpha-helix nucleation in two polyalanine-based peptides and in lysine and glutamic acid homopolymers was measured directly by stopped-flow deep UV CD with synchrotron radiation as the light source. Synchrotron radiation CD gives far superior signal to noise than a conventional instrument. The 16-aa AK peptide folds with first-order kinetics and a rate constant of 15 s-1 at 0 degrees C. The rate-determining step is presumably the initiation of a new helix, which occurs at least 10(5) times slower than expected. Helix folding occurs in at least two steps on the millisecond time scale for the longer peptides, with a transient overshoot of helix content significantly greater than at equilibrium, similar to that seen in the folding of several proteins. We suggest that the overshoot is caused by the formation of a single long helix followed by its breakage into the two or more helices present at equilibrium.

Side-chain structures in the first turn of the alpha-helix.

The first three residues at the N terminus of the alpha-helix are called N1, N2 and N3. We surveyed 2102 alpha-helix N termini in 298 high-resolution, non-homologous protein crystal structures for N1, N2 and N3 amino acid and side-chain rotamer propensities and hydrogen-bonding patterns. We find strong structural preferences that are unique to these sites. The rotamer distributions as a function of amino acid identity and position in the helix are often explained in terms of hydrogen-bonding interactions to the free N1, N2 and N3 backbone NH groups. Notably, the "good N2" amino acid residues Gln, Glu, Asp, Asn, Ser, Thr and His preferentially form i, i or i,i+1 hydrogen bonds to the backbone, though this is hindered by good N-caps (Asp, Asn, Ser, Thr and Cys) that compete for these hydrogen bond donors. We find a number of specific side-chain to side-chain interactions between N1 and N2 or between the N-cap and N2 or N3, such as Arg(N-cap) to Asp(N2). The strong energetic and structural preferences found for N1, N2 and N3, which differ greatly from positions within helix interiors, suggest that these sites should be treated explicitly in any consideration of helical structure in peptides or proteins.

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.