Radiative decay engineering: biophysical and biomedical applications.

Globular proteins fold by minimizing the nonpolar surface that is exposed to water, while simultaneously providing hydrogen-bonding interactions for buried backbone groups, usually in the form of secondary structures such as α-helices, β-sheets, and tight turns. A primary thermodynamic driving force for the formation of globular structure is thus the sequestration of nonpolar groups, but the correlation between the parts of proteins that are observed to fold first (termed folding initiation sites) and the “hydrophobicity” (as customarily defined) of the amino acids in these regions has been quite weak. It has previously been noted that many amino acid side chains contain considerable nonpolar sections, even if they also contain polar or charged groups. For example, a lysine side chain contains four methylenes, which may undergo hydrophobic interactions if the charged e-NH3+ group is salt-bridged or hydrogen-bonded. Folding initiation sites might therefore contain not only accepted “hydrophobic” amino acids, but also larger charged side chains. Recent experiments on the folding of mutant apomyoglobins provides corroboration for models based on the hypothesis that folding initiation sites arise from hydrophobic interactions. A near-perfect correlation was observed between the areas of the molecule that are present in the burst-phase kinetic intermediate and both the free energy of formation of hydrophobic initiation sites and the parameter “average area buried upon folding,” which pinpoints large side chains, even those containing charged or polar portions. These results provide a putative mechanism for the control of protein-folding initiation and growth by polar/nonpolar sequence propensity alone.

https://www.pnas.org/content/pnas/103/35/13057.full.pdf

The role of hydrophobic interactions in initiation and propagation of protein folding

Solute fluorescence quenching reactions were first applied to biochemical problems in the late 1960s and early 1970s, 7) and since that time they have been a very valuable research tool for studies with proteins, membranes, and other macromolecular assemblies. Quenching reactions are easy to perform, require only a small sample, usually are nondestructive, and can be applied to almost any system that has an intrinsic or extrinsic fluorescence probe. The most important characteristic, however, is the value of the information that these reactions can provide. Solute quenching reactions, using quenchers such as molecular oxygen, acrylamide, or iodide ion, provide information about the location of fluorescent groups in a macromolecular structure. A fluorophore that is located on the surface of a larger structure will be relatively accessible to a solute quencher that is dissolved in the aqueous phase. A fluorophore that is removed from the surface of a structure will be quenched to a lesser degree by the quencher. Thus, the quenching reaction can be used to probe topographical features of a macromolecular assembly and to sense any structural changes that may be caused by varying conditions or the addition of reagents. In addition, quenching reactions can, in some situations, provide information about conformational fluctuations. In Sections 2.3 and 2.4 I will discuss several examples of the use of solute quenchers in studies with proteins, membranes, and nucleic acids. Solute fluorescence quenching reactions can also be used to selectively alter the fluorescence properties of a sample in order to resolve contributions or aid in the measurement of data. To elaborate on this point, consider the different characteristics of fluorescence: the quantum yield, excitation and

Fluorescence Quenching: Theory and Applications

Optical sensing of analytes is often accomplished using fluorescence detection. Quantitation is usually performed by measuring the fluorescence of a probe whose intensity changes in response to the analyte of interest. In the present report we describe sensing based on changes in lifetime or decay time of the probe. Such a sensing scheme is advantageous because, in contrast to intensity measurements, the decay times are mostly independent of intensity changes due to light losses, photobleaching or probe washout. Present technology allows nanosecond (ns) decay times to be measured by the phase-modulation method with inexpensive and robust instrumentation. In particular, we describe lifetime-based pH sensing using a green 543 nm He-Ne laser, sensing of Ca2+ using 514 nm excitation and a Ca2+ competitive assay for glucose using an energy-transfer mechanism. We also discuss emerging applications of phase-modulation method sensing to cellular imaging and flow cytometry.

Fluorescence lifetime-based sensing of pH, Ca2+, K+ and glucose

Previous studies have demonstrated that His 12 plays a major role in the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here, amino acid replacement experiments show that His 12+ stabilizes the C-peptide helix chiefly by interacting with Phe 8. The Phe 8 ... His 12+ ring interaction is specific for the protonated form of His 12 (His 12+) and the interaction is not screened significantly by NaCl, unlike the charged group ... helix dipole interactions studied earlier in C-peptide. Analogs of C-peptide that are unable to form the Phe 8 ... His 12+ interaction show large increases in helix content for Phe----Ala and His----Ala. Therefore, the helical tendencies of the individual residues Phe, His, and Ala are important in determining the result of a replacement experiment. Since the side chains of Phe 8 and His 12 probably interact within the N-terminal helix of ribonuclease A, the existence of the Phe 8 ... His 12+ interaction in the isolated C-peptide helix adds to the evidence that the C-peptide helix is an autonomous folding unit.

/pdf/side-chain-interactions-in-the-c-peptide-helix-phe-8-his-12-4jw6nul6ju.pdf

Side-chain interactions in the C-peptide helix: Phe 8 ... His 12+.

Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.

Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol.

The C epsilon H proton resonance of His-12 of reduced cysteine S-sulfonated bovine pancreatic ribonuclease A exhibits a nonlinear temperature dependence of the chemical shift in its 1H-NMR spectrum at an apparent pH of 3.0. At temperatures below ca. 35 degrees C, the temperature dependence of the chemical shift of the His-12 C epsilon H resonance is opposite in sign to those of His-48, His-105, and His-119. At temperatures above ca. 35 degrees C, the temperature dependence of the chemical shift of the His-12 C epsilon H resonance is similar to those of the other three His C epsilon H resonances. These data indicate the existence of an equilibrium between locally ordered and locally disordered environments of His-12 in the sulfonated protein at temperatures below ca. 35 degrees C. The ordered and disordered conformations interconvert at a rate that is fast relative to the 1H-NMR chemical shift time scale--i.e., the locally ordered structure has a lifetime of much less than 7 msec. These results demonstrate that short- and medium-range interactions can define short-lived local structures under conditions of temperature and solution composition at which the native protein structure is stable. Furthermore, they demonstrate the utility of reduced derivatives of disulfide-containing proteins as model systems for the identification of local structures that may play a role as early-forming chain-folding initiation structures.

Local structure involving histidine-12 in reduced S-sulfonated ribonuclease A detected by proton NMR spectroscopy under folding conditions

Sodium sulfite as an antioxidant in the acid hydrolysis of bovine pancreatic ribonuclease A

Remarkable advances in molecular biology and biochemistry have sparked equally remarkable developments in the field of separations science. The use of cell culture for the production of genetically engineered proteins has made it possible to explore and directly intervene in the biochemical pathways that modulate disease. In recent years, we have seen the clinical successes of genetically engineered vaccines [1–5], anticancer agents [6,7], hormones [8], and thrombolytics [9,10]. 

Joel K. Swadesh

Papers

Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol.

Local structure involving histidine-12 in reduced S-sulfonated ribonuclease A detected by proton NMR spectroscopy under folding conditions

Sodium sulfite as an antioxidant in the acid hydrolysis of bovine pancreatic ribonuclease A

HPLC of Proteins and Peptides in the Pharmaceutical Industry