Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop.

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

Water irradiation devoid pulses enhance the sensitivity of 1H,1H nuclear Overhauser effects.

The nuclear Overhauser effect (NOE) is one of NMR spectroscopy's most important and versatile parameters. NOE is routinely utilized to determine the structures of medium-to-large size biomolecules and characterize protein-protein, protein-RNA, protein-DNA, and protein-ligand interactions in aqueous solutions. Typical [1H,1H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates 1H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons. However, at high- and ultra-high magnetic fields, the excitation of the water signal during the execution of the NOESY pulse sequences may cause significant attenuation of NOE cross-peak intensities. Using an evolutionary algorithm coupled with artificial intelligence, we recently designed high-fidelity pulses [Water irrAdiation DEvoid (WADE) pulses] that elude water excitation and irradiate broader bandwidths relative to commonly used pulses. Here, we demonstrate that WADE pulses, implemented into the 2D [1H,1H] NOESY experiments, increase the intensity of the NOE cross-peaks for labile and, to a lesser extent, non-exchangeable protons. We applied the new 2D [1H,1H] WADE-NOESY pulse sequence to two well-folded, medium-size proteins, i.e., the K48C mutant of ubiquitin and the Raf kinase inhibitor protein. We observed a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments. The new WADE pulses can be easily engineered into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences to boost their sensitivity.

Edmond Fischer's kinase legacy: History of the protein kinase inhibitor and protein kinase A.

Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cß subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

Allostery governs Cdk2 activation and differential recognition of CDK inhibitors.

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).

CHESPA/CHESCA-SPARKY: automated NMR data analysis plugins for SPARKY to map protein allostery.

MOTIVATION: Correlated Nuclear Magnetic Resonance (NMR) chemical shift changes identified through the CHEmical Shift Projection Analysis (CHESPA) and CHEmical Shift Covariance Analysis (CHESCA) reveal pathways of allosteric transitions in biological macromolecules. To address the need for an automated platform that implements CHESPA and CHESCA and integrates them with other NMR analysis software packages, we introduce here integrated plugins for NMRFAM-SPARKY that implement the seamless detection and visualization of allosteric networks. AVAILABILITY AND IMPLEMENTATION: CHESCA-SPARKY and CHESPA-SPARKY are available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). The assigned spectra involved in this study and tutorial videos using this dataset are available at https://sites.google.com/view/chescachespa-sparky. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Online.

Solid-State NMR of Membrane Proteins in Lipid Bilayers: To Spin or Not To Spin?

Membrane proteins mediate a plethora of cellular functions and represent important targets for drug development. Unlike soluble proteins, membrane proteins require native-like environments to fold correctly and be active. Therefore, modern structural biology techniques have aimed to determine the structure and dynamics of these membrane proteins at physiological temperature and in liquid crystalline lipid bilayers. With the flourishing of new NMR methodologies and improvements in sample preparations, magic angle spinning (MAS) and oriented sample solid-state NMR (OS-ssNMR) spectroscopy of membrane proteins is experiencing a new renaissance. Born as antagonistic approaches, these techniques nowadays offer complementary information on the structural topology and dynamics of membrane proteins reconstituted in lipid membranes. By spinning biosolid samples at the magic angle (θ = 54.7°), MAS NMR experiments remove the intrinsic anisotropy of the NMR interactions, increasing spectral resolution. Internuclear spin interactions (spin exchange) are reintroduced by RF pulses, providing distances and torsion angles to determine secondary, tertiary, and quaternary structures of membrane proteins. OS-ssNMR, on the other hand, directly detects anisotropic NMR parameters such as dipolar couplings (DC) and anisotropic chemical shifts (CS), providing orientational constraints to determine the architecture (i.e., topology) of membrane proteins relative to the lipid membrane. Defining the orientation of membrane proteins and their interactions with lipid membranes is of paramount importance since lipid-protein interactions can shape membrane protein conformations and ultimately define their functional states.In this Account, we report selected studies from our group integrating MAS and OS-ssNMR techniques to give a comprehensive view of the biological processes occurring at cellular membranes. We focus on the main experiments for both techniques, with an emphasis on new implementation to increase both sensitivity and spectral resolution. We also describe how the structural constraints derived from both isotropic and anisotropic NMR parameters are integrated into dynamic structural modeling using replica-averaged orientational-restrained molecular dynamics simulations (RAOR-MD). We showcase small membrane proteins that are involved in Ca2+ transport and regulate cardiac and skeletal muscle contractility: phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa). We summarize our results for the structures of these polypeptides free and in complex with the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA, 110 kDa). Additionally, we illustrate the progress toward the determination of the structural topology of a six transmembrane protein associated with succinate and acetate transport (SatP, hexamer 120 kDa). From these examples, the integrated MAS and OS-ssNMR approach, in combination with modern computational methods, emerges as a way to overcome the challenges posed by studying large membrane protein systems.

PHRONESIS: A One-Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid-State NMR Spectroscopy.

Solid-state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane-bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, 1 H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance-specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi-acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D 1 H-detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de-excitation of specific resonances were achieved by high-fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U-13 C,15 N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel 1 H-detected multi-acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.

Design and applications of water irradiation devoid RF pulses for ultra-high field biomolecular NMR spectroscopy.

Water suppression is of paramount importance for many biological and analytical NMR spectroscopy applications. Here, we report the design of a new set of binomial-like radio frequency (RF) pulses that elude water irradiation while exciting or refocusing the remainder of the 1H spectrum. These pulses were generated using a combination of an evolutionary algorithm and artificial intelligence. They display higher sensitivity relative to classical water suppression schemes and tunable water selectivity to avoid suppressing 1H resonances near the water signal. The broad bandwidth excitation obtained with these RF pulses makes them suitable for several NMR applications at high and ultra-high-field magnetic fields.

Dynamic allostery-based molecular workings of kinase:peptide complexes.

A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme:product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.

PISA-SPARKY: an interactive SPARKY plugin to analyze oriented solid-state NMR spectra of helical membrane proteins.

MOTIVATION: Two-dimensional [15N-1H] separated local field solid-state nuclear magnetic resonance (NMR) experiments of membrane proteins aligned in lipid bilayers provide tilt and rotation angles for α-helical segments using Polar Index Slant Angle (PISA)-wheel models. No integrated software has been made available for data analysis and visualization. RESULTS: We have developed the PISA-SPARKY plugin to seamlessly integrate PISA-wheel modeling into the NMRFAM-SPARKY platform. The plugin performs basic simulations, exhaustive fitting against experimental spectra, error analysis and dipolar and chemical shift wave plotting. The plugin also supports PyMOL integration and handling of parameters that describe variable alignment and dynamic scaling encountered with magnetically aligned media, ensuring optimal fitting and generation of restraints for structure calculation. AVAILABILITY AND IMPLEMENTATION: PISA-SPARKY is freely available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers of the SBGrid (https://sbgrid.org). The pisa.py script is available and documented on GitHub (https://github.com/weberdak/pisa.py) along with a tutorial video and sample data. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease.

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.

Molecular Mechanism for the Suppression of Alpha Synuclein Membrane Toxicity by an Unconventional Extracellular Chaperone.

Alpha synuclein (αS) oligomers are a key component of Lewy bodies implicated in Parkinson's disease (PD). Although primarily intracellular, extracellular αS exocytosed from neurons also contributes to PD pathogenesis through a prion-like transmission mechanism. Here, we show at progressive degrees of resolution that the most abundantly expressed extracellular protein, human serum albumin (HSA), inhibits αS oligomer (αSn) toxicity through a three-pronged mechanism. First, endogenous HSA targets αSn with sub-µM affinity via solvent-exposed hydrophobic sites, breaking the catalytic cycle that promotes αS self-association. Second, HSA remodels αS oligomers and high-MW fibrils into chimeric intermediates with reduced toxicity. Third, HSA unexpectedly suppresses membrane interactions with the N-terminal and central αS regions. Overall, our findings suggest that the extracellular proteostasis network may regulate αS cell-to-cell transmission not only by reducing the populations of membrane-binding competent αS oligomers but possibly also by shielding the membrane interface from residual toxic species.

Multi-receiver solid-state NMR using polarization optimized experiments (POE) at ultrafast magic angle spinning.

Ultrafast magic angle spinning (MAS) technology and 1H detection have dramatically enhanced the sensitivity of solid-state NMR (ssNMR) spectroscopy of biopolymers. We previously showed that, when combined with polarization optimized experiments (POE), these advancements enable the simultaneous acquisition of multi-dimensional 1H- or 13C-detected experiments using a single receiver. Here, we propose a new sub-class within the POE family, namely HC-DUMAS, HC-MEIOSIS, and HC-MAeSTOSO, that utilize dual receiver technology for the simultaneous detection of 1H and 13C nuclei. We also expand this approach to record 1H-, 13C-, and 15N-detected homonuclear 2D spectra simultaneously using three independent receivers. The combination of POE and multi-receiver technology will further shorten the total experimental time of ssNMR experiments for biological solids.

Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies.

Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

A theoretical assessment of structure determination of multi-span membrane proteins by oriented sample solid-state NMR spectroscopy.

Oriented sample solid state NMR (OS-ssNMR) spectroscopy allows direct determination of the structure and topology of membrane proteins reconstituted into aligned lipid bilayers. While OS-ssNMR theoretically has no upper size limit, its application to multi-span membrane proteins has not been established since most studies have been restricted to single or dual span proteins and peptides. Here, we present a critical assessment of the application of this method to multi-span membrane proteins. We used molecular dynamics simulations to back-calculate [15N-1H] separated local field (SLF) spectra from a G protein-coupled receptor (GPCR) and show that fully resolved spectra can be obtained theoretically for a multi-span membrane protein with currently achievable resonance linewidths.

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer.

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of "communities" composed of congruently dynamic residues that make up the protein kinase core. Girvan-Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.

Cytoplasmic nucleic acid-based XNAs directly enhance live cardiac cell function by a Ca2+ cycling-independent mechanism via the sarcomere.

Nucleic acid - protein interactions are critical for regulating gene activation in the nucleus. In the cytoplasm, however, potential nucleic acid-protein functional interactions are less clear. The emergence of a large and expanding number of non-coding RNAs and DNA fragments raises the possibility that the cytoplasmic nucleic acids may interact with cytoplasmic cellular components to directly alter key biological processes within the cell. We now show that both natural and synthetic nucleic acids, collectively XNAs, when introduced to the cytoplasm of live cell cardiac myocytes, markedly enhance contractile function via a mechanism that is independent of new translation, activation of the TLR-9 pathway or by altered intracellular Ca2+ cycling. Findings show a steep XNA oligo length-dependence, but not sequence dependence or nucleic acid moiety dependence, for cytoplasmic XNAs to hasten myocyte relaxation. XNAs localized to the sarcomere in a striated pattern and bound the cardiac troponin regulatory complex with high affinity in an electrostatic-dependent manner. Mechanistically, XNAs phenocopy PKA-based modified troponin to cause faster relaxation. Collectively, these data support a new role for cytoplasmic nucleic acids in directly modulating live cell cardiac performance and raise the possibility that cytoplasmic nucleic acid - protein interactions may alter functionally relevant pathways in other cell types.

Improving the quality of oriented membrane protein spectra using heat-compensated separated local field experiments.

Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is a powerful technique to determine the topology of membrane proteins in oriented lipid bilayers. Separated local field (SLF) experiments are central to this technique as they provide first-order orientational restraints, i.e., dipolar couplings and anisotropic chemical shifts. Despite the use of low-E (or E-free) probes, the heat generated during the execution of 2D and 3D SLF pulse sequences causes sizeable line-shape distortions. Here, we propose a new heat-compensated SE-SAMPI4 (hcSE-SAMPI4) pulse sequence that holds the temperature constant for the duration of the experiment. This modification of the SE-SAMPI4 results in sharper and more intense resonances without line-shape distortions. The spectral improvements are even more apparent when paramagnetic relaxation agents are used to speed up data collection. We tested the hcSE-SAMPI4 pulse sequence on a single-span membrane protein, sarcolipin (SLN), reconstituted in magnetically aligned lipid bicelles. In addition to eliminating peak distortions, the hcSE-SAMPI4 experiment increased the average signal-to-noise ratio by 20% with respect to the original SE-SAMPI4.

Hybridization of TEDOR and NCX MAS solid-state NMR experiments for simultaneous acquisition of heteronuclear correlation spectra and distance measurements.

Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is a major technique for the characterization of the structural dynamics of biopolymers at atomic resolution. However, the intrinsic low sensitivity of this technique poses significant limitations to its routine application in structural biology. Here we achieve substantial savings in experimental time using a new subclass of Polarization Optimized Experiments (POEs) that concatenate TEDOR and SPECIFIC-CP transfers into a single pulse sequence. Specifically, we designed new 2D and 3D experiments (2D TEDOR-NCX, 3D TEDOR-NCOCX, and 3D TEDOR-NCACX) to obtain distance measurements and heteronuclear chemical shift correlations for resonance assignments using only one experiment. We successfully tested these experiments on N-Acetyl-Val-Leu dipeptide, microcrystalline U-13C,15N ubiquitin, and single- and multi-span membrane proteins reconstituted in lipid membranes. These pulse sequences can be implemented on any ssNMR spectrometer equipped with standard solid-state hardware using only one receiver. Since these new POEs speed up data acquisition considerably, we anticipate their broad application to fibrillar, microcrystalline, and membrane-bound proteins.

More than a Liquid Junction: Effect of Stirring, Flow Rate, and Inward and Outward Electrolyte Diffusion on Reference Electrodes with Salt Bridges Contained in Nanoporous Glass.

The Henderson equation is usually used to calculate liquid-junction potentials between miscible electrolyte solutions. However, the potentials of reference electrodes that comprise an electrolyte-filled nanoporous glass frit may also be affected by charge screening. As reported previously, when the Debye length approaches or surpasses the glass pore diameter, reference potentials depend on the composition of the bridge electrolyte, the pore size of the frit, and the concentration of electrolyte in the sample. We report here that stirring of samples may alter the reference potential as it affects the electrolyte concentration in the section of the nanoporous glass frit that is facing the sample solution. When the flow rate of bridge electrolyte into the sample is small, convective mass transport of sample into the nanoporous frit occurs. The depth of penetration into the frit is only a few nanometers but, despite the use of concentrated salt bridges, this is enough to affect the extent of electrostatic screening when samples of low ionic strength are measured. Mixing of sample and salt bridge solutions-and in particular penetration of sample components into the frit-was optically monitored by observation of a deeply colored Fe[(SCN)(H2O)5]2+ complex that formed in situ exclusively in the region where the sample and salt bridge mixed. Importantly, because flow through nanoporous frits is very slow, mass transport through these frits is dominated by diffusion. Consequently, over as little as 1 h, reference electrode frits with low flow rates become contaminated with sample components and undergo depletion of electrolyte within the frit to a depth of several millimeters, which can negatively affect subsequent experiments.