Search PubMed⌕ Search

Biomedical subjects

G L Millhauser

Publications and source records attributed to G L Millhauser.

17 recordsLinked to original sources
PubMed1995

Exploring the peptide 3(10)-helix reversible alpha-helix equilibrium with double label electron spin resonance.

Over the last several years we have used spin labeling as a means for exploring the structure of helical peptides. Two nitroxide labels are engineered into a peptide sequence and distances are ranked with electron spin resonance (ESR). We have found that there is a significant amount of 3(10)-helix in 16-residue model peptides containing only L-amino acids. This review covers several facets of the methodology including spin labeling strategy, interpretation of ESR spectra and the influence of molecular dynamics on the spectral line shapes. Also covered are recent findings of a length-dependent 3(10)-helix-->alpha-helix transition and the role of Arg+ in the stabilization of specific helix structures.

Amino Acid Sequence↗
PubMed1994

A single carboxy-terminal arginine determines the amino-terminal helix conformation of an alanine-based peptide.

Arginine is a stabilizing element in both thermophilic and low molecular weight proteins. Similarly Lys+-->Arg+ substitutions increase the helix content of designed helical peptides. Here we explore this 'arginine effect' by examining how Lys+-->Arg+ substitutions influence the 3(10)-helix-->alpha-helix equilibrium in the helical peptide Ac-(AAAAK)3A-NH2. The unsubstituted sequence contains a significant amount of 3(10)-helix, however, single Lys+-->Arg+ substitutions shift the peptide conformation toward alpha-helix in a position-dependent fashion. The single substitution closest to the carboxy terminus induces the largest conformational change at the helix amino terminus. These findings suggest that a single strategically-placed arginine can exert long range control on helix structure.

Amino Acid Sequence↗
PubMed1993

Increasing sequence length favors alpha-helix over 3(10)-helix in alanine-based peptides: evidence for a length-dependent structural transition.

Ala-based peptides form marginally stable helices at low temperature and are conventionally considered as mixtures of alpha-helix and random coil. However, recent work with doubly spin-labeled peptides suggests that short 16-residue sequences contain a significant fraction of 3(10)-helix near the N-terminus (positions 4-8). Using the same double-label strategy, we report on the helix geometry of the peptides Ac-(AAAAK)nA-NH2 with n = 3 and n = 4. The 16-mer (n = 3) is now examined at a region near the C-terminus, and there is evidence for 3(10)-helix here as well. The 21-mer (n = 4) is examined in three regions of the sequence. In dramatic contrast to the 16-mer, the 21-mer exhibits the signature of alpha-helix at the N-terminus and on through the middle of the peptide. The 21-mer C-terminus, however, adopts the 3(10)-helix geometry as is often found for C-termini in protein alpha-helices. These data indicate that the proportion of alpha-helix and 3(10)-helix in Ala-based peptides depends upon the sequence length.

Alanine↗
PubMed1992

Short alanine-based peptides may form 3(10)-helices and not alpha-helices in aqueous solution.

Short alanine peptides, containing 16 or 17 residues, appear to form alpha-helices in aqueous solution. But the main spectroscopic analyses used on helical peptides (circular dichroism and nuclear magnetic resonance) cannot distinguish between an alpha-helix (in which the ith residue is hydrogen-bonded to residue i + 4; ref. 9) and the next most common peptide helix, the 3(10)-helix10 (i-->i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for alpha-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4-8), is inconsistent with an alpha-helical structure. Our experiments suggest that the more likely peptide geometry is a 3(10)-helix.

Alanine↗
PubMed1992

Rotational diffusion and intermolecular collisions of a spin labeled alpha-helical peptide determined by electron spin echo spectroscopy.

Short peptides that are composed mainly of alanine have recently been shown to form alpha-helices in aqueous solution at low temperature (Marqusee, S., and R. L. Baldwin. 1987. Proc. Natl. Acad. Sci. 84:8898-8902; Marqusee, S., V. H. Robbins, and R. L. Baldwin. 1989. Proc. Natl. Acad. Sci. USA. 86:5286-5290). These peptides are excellent models for probing structure and dynamics in isolated helical domains. In previous work we have designed and synthesized spin labeled analogs of these helix-forming peptides and we have shown that these analogs retain the folding characteristics of the parent peptide (Todd, A. P., and G. L. Millhauser. 1991. Biochemistry. 30:5515-5523). Using conventional continuous wave electron spin resonance (CW ESR) we have further shown that local motion is more pronounced near the helix amino terminus than in the central region as the peptide is thermally unfolded (Miick, S. M., A. P. Todd, and G. L. Millhauser. 1991. Biochemistry. 30:9498-9503). In this present work we use electron spin echo (ESE) spectroscopy to further refine our understanding of the solution dynamics of the 3K-8 peptide, which is a 16-mer with a nitroxide spin label attached at position 8. We find that the spin echo decays are well described by a single exponential function and that the determined correlation times are close to those previously derived from CW experiments. Variable concentration ESE experiments have directly revealed Heisenberg spin exchange (HSE) interactions and we find that the interpeptide collision rate is near to that expected for a free species in solution. This provides strong evidence that the helical conformation of these peptides is not stabilized by intermolecular interactions.

Alanine↗
PubMed1991

Position-dependent local motions in spin-labeled analogues of a short alpha-helical peptide determined by electron spin resonance.

We have used electron spin resonance and circular dichroism to examine and compare the dynamics in two analogues of the Ala-based 3K(I) peptide [Marqusee, S., Robbins, V.H., & Baldwin, R. L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290], labeled at positions 4 and 8, throughout the alpha-helix----coil transition. In the middle of the thermal unfolding transition, our results demonstrate that the local mobility near the N-terminus is greater than at the center of the peptide. This provides evidence, from the perspective of dynamics, that the ends of Ala-based alpha-helices are frayed. We further find that the position dependence of the mobility for the thermally unfolded state differs from that of the denaturant unfolded state. Only the latter state exhibits the local dynamics expected for a genuine random coil.

Alanine↗
PubMed1991

ESR spectra reflect local and global mobility in a short spin-labeled peptide throughout the alpha-helix----coil transition.

A series of short alanine-based synthetic peptides (16 or 17 residues) have previously been shown to exhibit an anomalously high degree of alpha-helicity [Marqusee, S., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290; Marqusee, S., & Baldwin, R.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8898-8902]. These peptides are ideal models for extracting position-dependent structural and dynamic information. Using the methanethiosulfonate nitroxide spin label (MTSSL), we labeled an analogue of the salt-bridge-stabilized "i+4" peptide, called the "i+4c", which has a specific attachment site created by replacing the central alanine with a cysteine. Circular dichroism (CD) spectra demonstrate that the i+4c-MTSSL peptide retains nearly the same helicity as the original i+4 peptide. The ESR spectra of the labeled peptide indicate no significant aggregation. ESR spectra were acquired throughout the helix-coil transition by temperature variation. From the motionally narrowed spectra, we extracted the rotational correlation times of the nitroxide label. Parallel measurements with circular dichroism enabled us to relate these parameters directly to the fractional helicity. For comparison, we followed a similar procedure with MTSSL-labeled glutathione (GS-MTSSL), a tripeptide that does not form an alpha-helix. Our results are interpreted in terms of a local tumbling volume, V(L), which reflects the portion of the peptide that reorients with the nitroxide label. At high fractional helicity, V(L) is similar to the volume expected for a 17-residue helix.

Amino Acid Sequence↗
PubMed1991

Diffusion model in ion channel gating. Extension to agonist-activated ion channels.

Previously, we described a model which treats ion channel gating as a discrete diffusion problem. In the case of agonist-activated channels at high agonist concentration, the model predicts that the closed lifetime probability density function from single channel recording approximates a power law with an exponent of -3/2 (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988a. Proc. Natl. Acad. Sci. USA. 85: 1503-1507). This prediction is consistent with distributions derived from a number of ligand-gated channels at high agonist concentration (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988b. Biophys. J. 54: 1165-1168.) but does not describe the behavior of ion channels at low activator concentrations. We examine here an extension of this model to include an agonist binding step. This extended model is consistent with the closed time distributions generated from the BC3H-1 nicotinic acetylcholine receptor for agonist concentrations varying over three orders of magnitude.

Animals↗
PubMed1990

Reptation theory of ion channel gating.

Reptation theory is a highly successful approach for describing polymer dynamics in entangled systems. In turn, this molecular process is the basis of viscoelasticity. We apply a modified version of reptation dynamics to develop an actual physical model of ion channel gating. We show that at times longer than microseconds these dynamics predict an alpha-helix-screw motion for the amphipathic protein segment that partially lines the channel pore. Such motion has been implicated in several molecular mechanics studies of both voltage-gated and transmitter-gated channels. The experimental probability density function (pdf) for this process follows t-3/2 which has been observed in several experimental systems. Reptation theory predicts that channel gating will occur on the millisecond time scale and this is consistent with experimental results from single-channel recording. We examine the consequences of reptation over random barriers and we show that, to first order, the pdf remains unchanged. In the case of a charged helix undergoing reptation in the presence of a transmembrane potential we show that the tail of the pdf will be exponential. We provide a list of practical experimental predictions to test the validity of this physical theory.

Elasticity↗
PubMed1988

A reevaluation of the mathematical models for simulating single-channel and whole-cell ionic currents.

We have developed a technique that allows for the simulation of both single-channel and whole-cell ionic currents given any arbitrary first-order kinetic scheme for the conformational states of an ion channel. The procedure is based on the solution of the master equation, which, in turn, is a general expression for a Markov process. The solution is expressed in terms of the eigenvalues and eigenvectors of the kinetic system and the system's deviation from equilibrium. Our derived expression provides a general recipe for the calculation of whole-cell currents. By further manipulation of this expression, we show how conditional probabilities are derived that can be used for the simulation of single-channel currents. We discuss computer implementation of the results so that complicated kinetic schemes can be solved numerically. Finally, we demonstrate the procedure by providing a worked example of a simple model of activation followed by inactivation.

Computer Simulation↗
PubMed1988

Rate-amplitude correlation from single-channel records. A hidden structure in ion channel gating kinetics?

Lifetime probability density functions from single-channel recording are usually assumed to follow a multiexponential form. The amplitudes and rate constants for each exponential component are presumed to be independent. We have explored this assumption and have found a correlation between the amplitudes and the rate constants in certain cases. We examine this correlation and the possibility that other functional forms may also properly describe these distribution functions.

Electric Conductivity↗
PubMed1988

Diffusion models of ion-channel gating and the origin of power-law distributions from single-channel recording.

The lifetimes of the unitary currents from ion channels, as revealed from single-channel recording, are traditionally thought to follow exponential or multiexponential distributions. The interpretation of these event-time distributions is that the gating process follows Markov kinetics among a small number of states. There is recent evidence, however, that certain systems exhibit distributions that follow power laws or functions related to power laws. Likewise, it has been suggested that data sets that appear to be multiexponential may be fit to simple power laws as well. In this paper we propose a different view of ion-channel-gating kinetics that is consistent with these recent experimental observations. We retain the Markovian nature of the kinetics, but, in contrast to the traditional models, we suggest that ion-channel proteins have a very large number of states all of similar energy. Gating, therefore, resembles a diffusion process. We show that our simplest one-dimensional model exhibits single-channel distributions that follow power laws of the form t-a, where 1/2 less than or equal to a less than or equal to 3/2. Exponents determined from recent experiments approximately fall within this range. We believe that this model is consistent with modern views of protein dynamics and, thus, may provide a key to the molecular details of the gating process.

Computer Simulation↗