Biomedical subjects

M S Kay

Publications and source records attributed to M S Kay.

9 recordsLinked to original sources

PubMed2001

Protein design of an HIV-1 entry inhibitor.

Human immunodeficiency virus type-1 (HIV-1) membrane fusion is promoted by the formation of a trimer-of-hairpins structure that brings the amino- and carboxyl-terminal regions of the gp41 envelope glycoprotein ectodomain into close proximity. Peptides derived from the carboxyl-terminal region (called C-peptides) potently inhibit HIV-1 entry by binding to the gp41 amino-terminal region. To test the converse of this inhibitory strategy, we designed a small protein, denoted 5-Helix, that binds the C-peptide region of gp41. The 5-Helix protein displays potent (nanomolar) inhibitory activity against diverse HIV-1 variants and may serve as the basis for a new class of antiviral agents. The inhibitory activity of 5-Helix also suggests a strategy for generating an HIV-1 neutralizing antibody response that targets the carboxyl-terminal region of the gp41 ectodomain.

Amino Acid Sequence↗

PubMed1999

Putative interhelix ion pairs involved in the stability of myoglobin.

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.

Animals↗

PubMed1999

Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate.

On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.

Amino Acid Sequence↗

PubMed1998

Alternative models for describing the acid unfolding of the apomyoglobin folding intermediate.

The acid-induced unfolding of the pH 4 intermediate of apomyoglobin (I) is described by either of two models: (1) a Monod-Wyman-Changeux-based model (MWC) where salt bridges perturb the pKa values of specific ionizable side chains, causing unfolding of I as these salt bridges are broken at low pH, and (2) the Linderstrom-Lang smeared charge model (L-L), which attributes acid unfolding of I to charge repulsion caused by the accumulation of positive charge on the surface of the protein. Both models fit earlier acid unfolding data well, but they make differing predictions about the effects of electrostatic mutants, which have been made and tested. Deletions of positive charge within I are found to stabilize I, but disruptions of potential salt bridges have little effect. These results show that the acid unfolding of I (I<-->U) is largely caused by generalized charge effects rather than by the loss of specific salt bridges. Acid unfolding of the native form, which is caused largely by a single histidine with a severely depressed pKa, is a sensitive indicator of changes in stability produced by mutations. In contrast, the I <--> U transition is caused by a number of groups with smaller pKa perturbations and both models predict that the pH midpoint of the I right harpoon over left harpoon U transition is an insensitive indicator of stability. This result reconciles previous conflicting results, in urea and acid unfolding studies of hydrophobic contact mutants, by showing that changes in the stability of I are poorly detected by acid unfolding.

Amino Acid Substitution↗

PubMed1998

KU activity: a method for calculating histopathologists' workloads.

An attempt to model activity of individual histopathologists or departments is illustrated, in which specimen numbers and types are weighted to produce an overall Kim unit (KU) activity. By cross referencing this numerical score against individual pathologists and clinicians, it is not only possible to interrogate departmental activity retrospectively, but also to predict and cost the impact of any change in service. The advantages over Welcans and Korner Units are illustrated.

Efficiency, Organizational↗

PubMed1997

Cooperativity of folding of the apomyoglobin pH 4 intermediate studied by glycine and proline mutations.

The apomyoglobin pH 4 folding intermediate contains the A, G, and H helices of myoglobin. Helix destabilizing mutations in the A and G helices are used to test whether the pH 4 folding intermediate of apomyoglobin folds cooperatively. Single glycine or proline mutations destabilize the intermediate substantially, showing that intrinsic helix propensities are important for stability of the intermediate. The A and G helices interact to stabilize each other, as shown by the effect of mutations in the G helix on the unfolding of the A helix, which can be monitored by tryptophan fluorescence. Wild type and the most stable mutant unfold in a two-state reaction, as shown by superposition of the unfolding curves measured by two probes (far-UV circular dichroism and Trp fluorescence), while unfolding of the less stable mutants is more complex. Cooperativity and stability of folding are linked also when stabilizing anions (sulphate, perchlorate) are used to adjust stability.

Animals↗

PubMed1996

Packing interactions in the apomyglobin folding intermediate.

The contribution of specific packing to the stability of the sperm whale apomyoglobin intermediate has been studied by urea denaturation monitored by circular dichroism and fluorescence. Mutations disrupting native packing sites within the subdomain formed by the A, G and H helices destabilize the intermediate, in contrast to the conclusion drawn from earlier studies of pH-induced unfolding. Based on these results, the intermediate is proposed to be stabilized by both partially formed native-like tertiary, and non-specific hydrophobic interactions forming a subdomain folding intermediate. The results help to explain how the intermediate acquires its structure and stability.

Acids↗

PubMed1995

Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway.

Apomyoglobin folding proceeds through a molten globule intermediate (low-salt form; I1) that has been characterized by equilibrium (pH 4) and kinetic (pH 6) folding experiments. Of the eight alpha-helices in myoglobin, three (A, G, and H) are structured in I1, while the rest appear to be unfolded. Here we report on the structure and stability of a second intermediate, the trichloroacetate form of the molten globule intermediate (I2), which is induced either from the acid-unfolded protein or from I1 by > or = 5 mM sodium trichloroacetate. Circular dichroism measurements monitoring urea- and acid-induced unfolding indicate that I2 is more highly structured and more stable than I1. Although I2 exhibits properties closer to those of the native protein, one-dimensional NMR spectra show that it maintains the lack of fixed side-chain structure that is the hallmark of a molten globule. Amide proton exchange and 1H-15N two-dimensional NMR experiments are used to identify the source of the extra helicity observed in I2. The results reveal that the existing A, G, and H helices present in I1 have become more stable in I2 and that a fourth helix--the B helix--has been incorporated into the molten globule. Available evidence is consistent with I2 being an on-pathway intermediate. The data support the view that apomyoglobin folds in a sequential fashion through a single pathway populated by intermediates of increasing structure and stability.

Apoproteins↗

PubMed1991

U-3'-BCIP: a chromogenic substrate for the detection of RNase A in recombinant DNA expression systems.

The synthesis of the bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5) chromogenic substrate uridine-3'-(5-bromo-4-chloroindol-3-yl)-phosphate (U-3'-BCIP) is described. RNase A catalyzes the hydrolysis of U-3'-BCIP to release a halogenated indol-3-ol that undergoes rapid aerobic oxidation to the dark blue 5,5'-dibromo-4,4'-dichloroindigo. Preliminary kinetic studies indicate that this compound may have practical use for assaying RNase A activity both in vitro and in vivo, e.g. in screening bacterial colonies for RNase A produced by recombinant DNA methods.

Animals↗