Zhengshuang Shi

Polyproline II structure in a sequence of seven alanine residues

A sequence of seven alanine residues—too short to form an α-helix and whose side chains do not interact with each other—is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The 3JHNα coupling constants of individual alanine residues have been measured from 2 to 56°C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2°C and the content of β strand is increased by approximately 10% at 55°C relative to that at 2°C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer “random coil” peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue.

Is polyproline II a major backbone conformation in unfolded proteins

Publisher Summary Protein folding is a process by which a polypeptide chain acquires its native structure from an unfolded state through a transition state. Recent studies of the unfolded states of proteins are based on a modification of the random coil model, recognizing that in many cases some residual native or non-native structure persists.. Combined evidence from the theoretical study of a blocked alanine peptide in aqueous solution and a variety of spectroscopic studies, including ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), two-dimensional vibrational spectroscopy, vibrational circular dichroism (VCD), and vibrational Raman optical activity (VROA) reveal that the polyproline II (P II ) conformation is the dominant conformation in a variety of short model peptides. This chapter discusses the evidence from short peptides. It reviews the circular dichroism of unfolded proteins and addresses the role of P II in unfolded proteins.

Cooperative helix stabilization by complex Arg–Glu salt bridges

Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg+–Glu−–Arg+ spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins. Proteins 2001;44:123–132.

Stabilization of α‐helix structure by polar side‐chain interactions: Complex salt bridges, cation–π interactions, and C–H … O H‐bonds

It is generally understood that helical proteins are stabilized by a combination of hydrophobic and packing interactions, together with H-bonds and electrostatic interactions. Here we show that polar side-chain interactions on the surface can play an important role in helix formation and stability. We review studies on model helical peptides that reveal the effect of weak interactions between side chains on helix stability, focusing on some nonclassical side-chain-side-chain interactions: complex salt bridges, cation-pi, and C-H em leader O H-bonding interactions. Each of these can be shown to contribute to helix stability, and thus must be included in a comprehensive catalogue of helix stabilizing effects. The issue of the structure of the unfolded states of helical peptides is also discussed, in the light of recent experiments showing that these contain substantial amounts of polyproline II conformation.

Antimicrobial dendrimer active against Escherichia coli biofilms

We have investigated the ability of a previously reported antimicrobial peptide dendrimer (RW)(4D) to inactivate Escherichia coli RP437 in planktonic culture and in biofilms. The results show that the dendrimer inhibits bacterial growth in both planktonic and biofilm states. Live/Dead staining assays reveal that most bacteria in a preformed biofilm lose viability after treatment with this peptide. This result is in marked contrast to most existing reports that antimicrobial peptides are ineffective against mature bacterial biofilms.

Non-classical helix-stabilizing interactions: CH⋯O H-bonding between Phe and Glu side chains in α-helical peptides

The classical picture of H-bonds has evolved considerably. In contrast to earlier expectations, C-H...O H-bonds are now known to be prevalent in both small organic and large biological systems. However, there are few reports on the energetic contribution of C-H...O H-bonds in protein or polypeptide systems and we do not know whether such interactions are stabilizing. Here we investigate C-H...O H-bonding interactions between Phe and Glu side chains by determining their effects on the helicity of model alpha-helical peptides using a combination of CD and NMR spectroscopy. The results suggest that Glu/Phe C-H...O H-bonding interactions stabilize helical structure, but only in the orientation Glu --> Phe (N --> C). Each Glu --> Phe (N --> C) interaction can contribute approximately -0.5 kcal mol(-1) to the stability of helical peptide. In the reverse orientation, Phe --> Glu (N --> C) appears to contribute negligibly. pH titrations provide further evidence for the existence of C-H...O H-bonds. The C-H...O H-bonding interactions in these peptides are insensitive to the screening effect of added neutral salt. Our results provide quantitative energetic information on C-H...O H-bonds that should be useful for empirical force-field calibration.

Structure and antimicrobial properties of multivalent short peptides

The emergence of multidrug-resistant (MDR) strains continues to threaten human health. Along with many efforts directed towards the development of antimicrobial peptides (AMPs) as potential antibiotics, we have designed and tested several series of multivalent AMPs in comparison with the natural AMP, indolicidin. Constructs include multivalent displays with different sequences, repeats and scaffolds, including dendrimers, brush-like structures and polymeric displays constructed by linking various peptides to polymaleic anhydride (PMA). A branched tetramer of dipeptides (RW)4D demonstrates the highest level of effectiveness among the constructs of this study. The results also show that under extended exposure to sublethal doses of (RW)4D and indolicidin over a 400 generations period, MDR strains elicit much lower levels of resistance than to traditional antibiotics or disinfectants such as ciprofloxacin, vancomycin, chlorhexidine and gentamicin.

PII structure in the model peptides for unfolded proteins: Studies on ubiquitin fragments and several alanine‐rich peptides containing QQQ, SSS, FFF, and VVV

A great deal of attention has been paid lately to the structures in unfolded proteins due to the recent discovery of many biologically functional but natively unfolded proteins and the far‐reaching implications of order in unfolded states for protein folding. Recently, studies on oligo‐Ala, oligo‐Lys, oligo‐Asp, and oligo‐Glu, as well as oligo‐Pro, have indicated that the left‐handed polyproline II (PII) is the major local structure in these short peptides. Here, we show by NMR and CD studies that ubiquitin fragments, model unfolded peptides composed of nonrepeating amino acids, and four alanine‐rich peptides containing QQQ, SSS, FFF, and VVV sequences are all present in aqueous solution predominantly in the extended PII or β conformation. The results from this and related studies indicate that PII might be a major backbone conformation in unfolded proteins. The presence of defined local backbone structure in unfolded proteins is inconsistent with predictions from random coil models. Proteins 2006.

Ramachandran redux

Protein molecules are indispensable to life processes, ranging from catalysis of reactions to transport, signaling, and shaping of cells (1). Despite their intricate architecture, revealed in thousands of 3D structures stored in the Protein Data Bank (2), protein structures rest on a surprisingly small set of principles (1). Perhaps most fundamental of all is the fact that the amide bond (Fig. 1A) is planar, so that only two dihedral angles, denoted by Φ and Ψ (Fig. 1A), define the conformation of the bond linking adjacent amino acids. Following leads from their studies of the structure of collagen, the predominant protein in humans, the crystallographer G. N. Ramachandran and his colleagues first used a 2D diagram to depict the geometry of a dipeptide—two amino acids together with the intervening amide bond (3). They plotted values of one of the angles along the x axis and the second along the y axis, as shown in Fig. 1B. Using the few peptide structures then available, they could see that the angles clustered in only a few sections of the map. Model building led them to conclude that most values of the two angles were inaccessible owing to collisions between atoms of the backbone (Fig. 1B). It is hard to overstate the seminal impact of this representation, which has guided the thinking of protein chemists and structural biologists for many decades, as pointed out by Porter and Rose (4). The article in PNAS by Porter and Rose (4) reports on a unique and quite surprising aspect of the structure of proteins. They have refined the classic Ramachandran plot …

OH radical production stimulated by (RW)4D, a synthetic antimicrobial agent and indolicidin

Antimicrobial peptides (AMPs) have been widely studied as a potential class of antibiotics for treating infectious diseases caused by multidrug-resistant (MDR) bacterial strains. While the cell surface and membrane are generally implicated in the antibacterial action of AMPs, we still lack a complete understanding of the detailed mechanism(s) involved. Here we show that the natural antimicrobial peptide indolicidin (ILN) and a more potent synthetic dendrimeric analog, (RW)4D, generate hydroxide radicals in target bacterial cells via a Fenton reaction, as Collins group has reported for bactericidal antibiotics. Our results further suggest that the TCA cycle does not regulate release of irons from iron clusters, and electron donors such as NADH from the TCA cycle are only partially responsible for superoxide generation. Since AMPs are refractory to bacterial resistance, unlike the bacteriocidal antibiotics tested, we hypothesize that the source of the OH radicals differs from that in the latter case.