Cytotoxicity of RNase Sa to the acute myeloid leukemia Kasumi-1 cells depends on the net charge.

The majority of known cytotoxic RNases are basic proteins which destroy intracellular RNA. Cationization of RNases is considered to be an effective strategy for strengthening their antitumor properties. We constructed a set of RNase Sa variants consisting of charge reversal mutants, charge neutralization mutants, and variants with positively charged cluster at the N-terminus.

Forces stabilizing proteins.

The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a -CH2- group on folding contributes 1.

Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A.

Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility.

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins.

Contribution of hydrophobic interactions to protein stability.

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.

Audio-visual distraction during spinal anaesthesia for orthopaedic surgery.

Spinal anaesthesia is an effective method of delivering anaesthesia for primary total knee replacement. A regional technique has several benefits over an opioid based general anaesthetic (Fischer 2009) (Figure 1). Pre-medication and sedation is commonly used as an adjunct to create anxiolysis and reduce stress which could be provoked by being awake in the theatre environment.

Increasing protein stability: importance of DeltaC(p) and the denatured state.

Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the beta-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa.

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins.

The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure.

Factors that influence helical preferences for singly charged gas-phase peptide ions: the effects of multiple potential charge-carrying sites.

Ion mobility-mass spectrometry is used to investigate the structure(s) of a series of model peptide [M + H](+) ions to better understand how intrinsic properties affect structure in low dielectric environments. The influence of peptide length, amino acid sequence, and composition on gas-phase structure is examined for a series of model peptides that have been previously studied in solution. Collision cross sections for the [M + H](+) ions of Ac-(AAKAA)(n)Y-NH(2) (n = 3-6) and Ac-Y(AEAAKA)(n)F-NH(2) (n = 2-5) are reported and correlated with candidate structures generated using molecular modeling techniques.

Increasing protein stability by improving beta-turns.

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions.

A summary of the measured pK values of the ionizable groups in folded proteins.

We tabulated 541 measured pK values reported in the literature for the Asp, Glu, His, Cys, Tyr, and Lys side chains, and the C and N termini of 78 folded proteins. The majority of these values are for the Asp, Glu, and His side chains. The average pK values are Asp 3.

Protein ionizable groups: pK values and their contribution to protein stability and solubility.

The structure, stability, solubility, and function of proteins depend on their net charge and on the ionization state of the individual residues. Consequently, biochemists are interested in the pK values of the ionizable groups in proteins and how these pK values depend on their environment. We review what has been learned about pK values of ionizable groups in proteins from experimental studies and discuss the important contributions they make to protein stability and solubility.

Determining the conformational stability of a protein using urea denaturation curves.

The stability of globular proteins is an important factor in determining their usefulness in basic research and medicine. A number of environmental factors contribute to the conformational stability of a protein, including pH, temperature, and ionic strength. In addition, variants of proteins may show remarkable differences in stability from their wild-type form.

Solvent denaturation of proteins and interpretations of the m value.

The stability of globular proteins is important in medicine, proteomics, and basic research. The conformational stability of the folded state can be determined experimentally by analyzing urea, guanidinium chloride, and thermal denaturation curves. Solvent denaturation curves in particular may give useful information about a protein such as the existence of domains or the presence of stable folding intermediates.

Spectrophotometric determination of protein concentration.

The concentration of a purified protein in solution is most conveniently and accurately measured using absorbance spectroscopy. The absorbance, A, is a linear function of the molar concentration, C, according to the Beer-Lambert law: A = epsilon x l x c, where e is the molar absorption coefficient and l is the cell path length. This unit provides protocols for calculation of epsilon for a folded or unfolded protein, making use of the average epsilon values for the three contributing chromophores in proteins (the side chains of Trp, Tyr, and Cys).

RNase-induced apoptosis: fate of calcium-activated potassium channels.

The connection between the action of microbial RNases and Ca2+-activated K+ (KCa) channels was investigated in human embryo kidney cells HEKhSK4 artificially expressing the channels. These channels protected HEKhSK4 cells from apoptosis induced by binase and 5K charge reversal mutant of RNase Sa. After the first 24h, potassium current increased without increase in intracellular Ca2+, and mitochondrial potential remained high.

Measuring and increasing protein solubility.

High concentration protein delivery is difficult to achieve for several protein pharmaceuticals due to low solubility. In this review, we discuss different types of low protein solubility, including low in vitro solubility, which is relevant to the formulation of protein pharmaceuticals. We also discuss different methods of measuring protein solubility with an emphasis on the method of inducing amorphous precipitation using ammonium sulfate.

Peptide sequence and conformation strongly influence tryptophan fluorescence.

This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG.

Tryptophan fluorescence reveals the presence of long-range interactions in the denatured state of ribonuclease Sa.

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues.

Increasing protein conformational stability by optimizing beta-turn sequence.

Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions.

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa.

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done.

Hydrogen bonding markedly reduces the pK of buried carboxyl groups in proteins.

The ionizable groups in proteins with the lowest pKs are the carboxyl groups of aspartic acid side-chains. One of the lowest, pK=0.6, is observed for Asp76 in ribonuclease T1.

pK values of the ionizable groups of proteins.

We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.