Benjamin H. McMahon

Slaving: Solvent fluctuations dominate protein dynamics and functions

Protein motions are essential for function. Comparing protein processes with the dielectric fluctuations of the surrounding solvent shows that they fall into two classes: nonslaved and slaved. Nonslaved processes are independent of the solvent motions; their rates are determined by the protein conformation and vibrational dynamics. Slaved processes are tightly coupled to the solvent; their rates have approximately the same temperature dependence as the rate of the solvent fluctuations, but they are smaller. Because the temperature dependence is determined by the activation enthalpy, we propose that the solvent is responsible for the activation enthalpy, whereas the protein and the hydration shell control the activation entropy through the energy landscape. Bond formation is the prototype of nonslaved processes; opening and closing of channels are quintessential slaved motions. The prevalence of slaved motions highlights the importance of the environment in cells and membranes for the function of proteins.

A unified model of protein dynamics

Protein functions require conformational motions. We show here that the dominant conformational motions are slaved by the hydration shell and the bulk solvent. The protein contributes the structure necessary for function. We formulate a model that is based on experiments, insights from the physics of glass-forming liquids, and the concepts of a hierarchically organized energy landscape. To explore the effect of external fluctuations on protein dynamics, we measure the fluctuations in the bulk solvent and the hydration shell with broadband dielectric spectroscopy and compare them with internal fluctuations measured with the Mössbauer effect and neutron scattering. The result is clear. Large-scale protein motions are slaved to the fluctuations in the bulk solvent. They are controlled by the solvent viscosity, and are absent in a solid environment. Internal protein motions are slaved to the beta fluctuations of the hydration shell, are controlled by hydration, and are absent in a dehydrated protein. The model quantitatively predicts the rapid increase of the mean-square displacement above ≈200 K, shows that the external beta fluctuations determine the temperature- and time-dependence of the passage of carbon monoxide through myoglobin, and explains the nonexponential time dependence of the protein relaxation after photodissociation.

The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin.

The grail of protein science is the connection between structure and function. For myoglobin (Mb) this goal is close. Described as only a passive dioxygen storage protein in texts, we argue here that Mb is actually an allosteric enzyme that can catalyze reactions among small molecules. Studies of the structural, spectroscopic, and kinetic properties of Mb lead to a model that relates structure, energy landscape, dynamics, and function. Mb functions as a miniature chemical reactor, concentrating and orienting diatomic molecules such as NO, CO, O2, and H2O2 in highly conserved internal cavities. Reactions can be controlled because Mb exists in distinct taxonomic substates with different catalytic properties and connectivities of internal cavities.

Protein folding is slaved to solvent motions

Proteins, the workhorses of living systems, are constructed from chains of amino acids, which are synthesized in the cell based on the instructions of the genetic code and then folded into working proteins. The time for folding varies from microseconds to hours. What controls the folding rate is hotly debated. We postulate here that folding has the same temperature dependence as the α-fluctuations in the bulk solvent but is much slower. We call this behavior slaving. Slaving has been observed in folded proteins: Large-scale protein motions follow the solvent fluctuations with rate coefficient kα but can be slower by a large factor. Slowing occurs because large-scale motions proceed in many small steps, each determined by kα. If conformational motions of folded proteins are slaved, so a fortiori must be the motions during folding. The unfolded protein makes a Brownian walk in the conformational space to the folded structure, with each step controlled by kα. Because the number of conformational substates in the unfolded protein is extremely large, the folding rate coefficient, kf, is much smaller than kα. The slaving model implies that the activation enthalpy of folding is dominated by the solvent, whereas the number of steps nf = kα/kf is controlled by the number of accessible substates in the unfolded protein and the solvent. Proteins, however, undergo not only α- but also β-fluctuations. These additional fluctuations are local protein motions that are essentially independent of the bulk solvent fluctuations and may be relevant at late stages of folding.

Myoglobin: the hydrogen atom of biology and a paradigm of complexity.

In a tour de force in this issue of PNAS, Bourgeois et al. (1) have used 2.2-ns x-ray pulses to observe the motion of carbon monoxide (CO) through myoglobin (Mb) and the relaxation of the protein from 3.2 ns to 3 ms after photodissociation. This work follows the pioneering experiments of Moffat and collaborators (2). It demonstrates how far advances in x-ray sources and computers have moved the field of protein structure determination since the path-breaking work of Kendrew et al . and Perutz (3, 4). The recent breakthrough shows how careful studies of proteins, in particular of Mb, impact many different fields. Mb is a monomeric protein that gives muscle its red color. Thirty years ago the textbook function of Mb, storage of dioxygen at the heme iron, was considered to be simple, fully understood, and consequently boring. Mb was essentially written off as a topic of serious research. Since then, the situation has changed: Mb is no longer fully understood. It plays roles other than O2 storage, serves as a prototype for complex systems, and yields insight into the chemistry and physics of soft matter and of chemical reactions. Mb consists of 153 amino acids that fold into a structure that is ≈3 nm in diameter, as depicted in Fig. 1 a . Fig. 1 b gives a schematic cross section through Mb that shows the active center: a heme group with a central iron atom. Surrounding the heme group are five cavities, the heme cavity and four cavities denoted by Xe1 to Xe4 (5). The amino acids lining the xenon cavities are much more conserved than other amino acids in mammalian Mb; they are thus likely to be important for function. A major part of Mb is taken up by amino acids that do not appear to …

Hydration, slaving and protein function

Protein dynamics is crucial for protein function. Proteins in living systems are not isolated, but operate in networks and in a carefully regulated environment. Understanding the external control of protein dynamics is consequently important. Hydration and solvent viscosity are among the salient properties of the environment. Dehydrated proteins and proteins in a rigid environment do not function properly. It is consequently important to understand the effect of hydration and solvent viscosity in detail. We discuss experiments that separate the two effects. These experiments have predominantly been performed with wild-type horse and sperm whale myoglobin, using the binding of carbon monoxide over a broad range of temperatures as a tool. The experiments demonstrate that data taken only in the physiological temperature range are not sufficient to understand the effect of hydration and solvent on protein relaxation and function. While the actual data come from myoglobin, it is expected that the results apply to most or all globular proteins.

Microscopic model of carbon monoxide binding to myoglobin

We present a microscopic model of carbon monoxide (CO) binding to myoglobin which reproduces the experimentally observed Arrhenius pre-exponential factor of 109 s−1 and activation enthalpy distribution centered at 12 kJ/mol. The model is based on extensive ab initio calculations of CO interacting with a model heme-imidazole group which we performed using a fully quantum mechanical Hartree–Fock/density functional theory (HF/DFT) hybrid method. We fit the HF/DFT calculated energies, obtained for over 1000 heme-CO structures with varied CO and iron positions and orientations for both high (S=2) and low (S=0) spin states, to a model potential function which includes a bonding interaction in both of the spin states, electrostatic, and anisotropic Lennard-Jones-type interactions. By combining the x-ray determined protein structure with this potential and protein-CO interactions and internal heme interaction potentials obtained from established molecular dynamics literature, we calculate the energy required for ...

Rapid phylogenetic and functional classification of short genomic fragments with signature peptides

BackgroundClassification is difficult for shotgun metagenomics data from environments such as soils, where the diversity of sequences is high and where reference sequences from close relatives may not exist. Approaches based on sequence-similarity scores must deal with the confounding effects that inheritance and functional pressures exert on the relation between scores and phylogenetic distance, while approaches based on sequence alignment and tree-building are typically limited to a small fraction of gene families. We describe an approach based on finding one or more exact matches between a read and a precomputed set of peptide 10-mers.ResultsAt even the largest phylogenetic distances, thousands of 10-mer peptide exact matches can be found between pairs of bacterial genomes. Genes that share one or more peptide 10-mers typically have high reciprocal BLAST scores. Among a set of 403 representative bacterial genomes, some 20 million 10-mer peptides were found to be shared. We assign each of these peptides as a signature of a particular node in a phylogenetic reference tree based on the RNA polymerase genes. We classify the phylogeny of a genomic fragment (e.g., read) at the most specific node on the reference tree that is consistent with the phylogeny of observed signature peptides it contains. Using both synthetic data from four newly-sequenced soil-bacterium genomes and ten real soil metagenomics data sets, we demonstrate a sensitivity and specificity comparable to that of the MEGAN metagenomics analysis package using BLASTX against the NR database. Phylogenetic and functional similarity metrics applied to real metagenomics data indicates a signal-to-noise ratio of approximately 400 for distinguishing among environments. Our method assigns ~6.6 Gbp/hr on a single CPU, compared with 25 kbp/hr for methods based on BLASTX against the NR database.ConclusionsClassification by exact matching against a precomputed list of signature peptides provides comparable results to existing techniques for reads longer than about 300 bp and does not degrade severely with shorter reads. Orders of magnitude faster than existing methods, the approach is suitable now for inclusion in analysis pipelines and appears to be extensible in several different directions.

U.S. airport entry screening in response to pandemic influenza: modeling and analysis.

Summary Background A stochastic discrete event simulation model was developed to assess the effectiveness of passenger screening for Pandemic Influenza (PI) at U.S. airport foreign entry. Methods International passengers arriving at 18 U.S. airports from Asia, Europe, South America, and Canada were assigned to one of three states: not infected, infected with PI, infected with other respiratory illness. Passengers passed through layered screening then exited the model. 80% screening effectiveness was assumed for symptomatic passengers; 6% asymptomatic passengers. Results In the first 100 days of a global pandemic, U.S. airport screening would evaluate over 17M passengers with 800K secondary screenings. 11,570 PI infected passengers (majority asymptomatic) would enter the U.S. undetected from all 18 airports. Foreign airport departure screening significantly decreased the false negative (infected/undetected) passengers. U.S. attack rates: no screening (26.9%–30.9%); screening (26.4%–30.6%); however airport screening results in 800K–1.8M less U.S. PI cases; 16K–35K less deaths (2% fatality rate). Antiviral medications for travel contact prophylaxis (10 contacts/PI passenger) were high – 8.8M. False positives from all 18 airports: 100–200/day. Conclusions Foreign shore exit screening greatly reduces numbers of PI infected passengers. U.S. airport screening identifies 50% infected individuals; efficacy is limited by the asymptomatic PI infected. Screening will not significantly delay arrival of PI via international air transport, but will reduce the rate of new US cases and subsequent deaths.

Conformational dependence of a protein kinase phosphate transfer reaction

Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase are calculated by plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. With the protein in TC, the motions involved in reaction are small, with only Pγ and the catalytic proton moving >0.5 Å. Examination of the structures reveals that in the RC the active site cleft is not completely closed and there is insufficient space for the phosphorylated serine residue in the product state. Together, these observations imply that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site.